Systems and methods for rapidly identifying useful chemicals in liquid samples

ABSTRACT

The present invention provides for systems and methods that utilize automated and integratable workstations for identifying chemicals having useful activity. The present invention is also directed to chemical entities and information (e.g., chemical or biological activities of chemicals) generated or discovered by operation of workstations of the present invention. The present invention includes automated workstations that are programmably controlled to minimize processing times at each workstation and that can be integrated to minimize the processing time of the liquid samples from the start to finish of the process.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.08/858,016, filed on May 16. 1997, entitled “Systems and Methods forRapidly Identifying Useful Chemicals in Liquid Samples” and claimspriority thereto under 35 U.S.C. § 120. The content of the 08/858,016now U.S. Pat. No. 5,985,214 application is hereby incorporated byreference in its entirety.

TECHNICAL FIELD

The present invention generally relates to automated and integratedsystems and methods for rapidly identifying chemicals with biologicalactivity in liquid samples, particularly automated screening of lowvolume samples for new medicines, agrochemicals, or cosmetics.

BACKGROUND

Systems and methods for rapidly identifying chemicals with biologicalactivity in samples, especially small liquid samples, can benefit anumber of different fields. For instance, the agrochemical,pharmaceutical, and cosmetic fields all have applications where largenumbers of liquid samples containing chemicals are processed. Currently,many such fields use various strategies to reduce processing times, suchas simplified chemistry, semi-automation and robotics. While suchstrategies may improve the processing time for a particular type ofliquid sample, process step or chemical reaction, such methods orapparatuses can seldom integrate the entire process, especially thegeneration or detection of chemical events in small volumes. Suchapparatuses are also often limited in their application, since many ofthem are designed for, and dedicated to, a particular type of liquidsample or chemical reaction.

In most processes involving liquid samples, as the complexity of theliquid sample processing increases the process time per sampleincreases. Although, some very simple chemical reactions or liquidprocessing methods can achieve extremely high throughput rates, such asin the manufacturing of containerized liquids, complicated processing ofliquids is typically several orders of magnitude slower. In someinstances, the processing of liquid samples, such as in pharmaceuticalarts, which usually demands complicated liquid processing for drugdiscovery, can obtain throughput rates of approximately 3,000 samplesper day. This type of processing in general, however, uses liquid samplevolumes on the order of 100 to 200 microliters, which often requiresrelatively large amounts of exotic and expensive reagents, and does nottypically incorporate automated access to large stores of liquidreagents.

Consequently, there is a need to provide components, systems and methodsfor rapidly processing liquid samples at high throughput rates,particularly liquid samples of microliter volumes, one to tenmicroliters, to identify chemicals with useful activity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows one embodiment of a liquid sample processing system andexemplary routes of information and plate transfer within.

FIG. 2 shows one embodiment of a liquid sample processing system thatcould be used for identifying useful chemicals and exemplary routes ofinformation and plate transfer.

FIG. 3 shows one embodiment of a storage and retrieval module.

FIG. 4 shows one embodiment of a sample distribution module.

FIG. 5 shows one embodiment of a screening system.

FIGS. 6A and B show embodiments of a workstation interface.

FIG. 7 shows one embodiment of a reagent dispensor.

FIG. 8 shows one embodiment of a detector.

FIG. 9 shows one embodiment of a data structure.

FIG. 10A shows one embodiment of a user interface.

FIG. 10B shows one embodiment of a user interface.

FIG. 11 shows one embodiment of a screening system.

FIG. 12 shows one embodiment of screening system material flow.

FIG. 13 shows one embodiment of an user interaction flowchart.

FIG. 14 shows one embodiment of a storage and retrieval end actuator.

FIGS. 15A and B shows one embodiment of a universal stacker.

FIGS. 16A and B shows one embodiment of transport system elements.

FIG. 17 shows one embodiment of a hit profiling workstation.

FIG. 18 shows different dispensing positions.

FIGS. 19A and B shows one embodiment of a screening reagent dispensingrobot.

SUMMARY

The present invention is directed to systems and methods that utilizeautomated and integratable workstations for identifying chemicals havinguseful activity. The present invention is also directed to chemicalentities and information (e.g., chemical or biological activities ofchemicals) generated or discovered by operation of workstations of thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Generally, the nomenclatureused herein and many of the automation, computer, detection, chemistryand laboratory procedures described below are those well known andcommonly employed in the art. Standard techniques are usually used forengineering, robotics, informatics, optics, molecular biology, computersoftware and integration. Generally, chemical reactions, cell assays andenzymatic reactions are performed according to the manufacturer'sspecifications where appropriate. The techniques and procedures aregenerally performed according to conventional methods in the art andvarious general references (see generally, Knuth, Donald E., The Art ofComputer Propramming, Volume 1, Fundamental Algorithms, Third Edition(Reading, Massachusetts: Addison-Wesley, 1997); Volume 2, SeminumericalAlgorithms, Second Edition (Reading, Massachusetts: Addison-Wesley,1981); Volume 3, Sorting and Searching, (Reading, Massachusetts:Addison-Wesley, 1973); for computational methods, Lakowicz, J. R.Principles of Fluorescence Spectroscopy, New York: Plenum Press (1983)for fluorescence techniques and Sambrook et al. Molecular Cloning: ALaboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. for molecular biology methods, which areincorporated herein by reference which are provided throughout thisdocument). The nomenclature used herein and the laboratory procedures inchemistry, molecular biology, automation, computer sciences, and drugdiscovery described below are those well known and commonly employed inthe art. Standard techniques are often used for chemical syntheses,chemical analyses, drug screening, and diagnosis. As employed throughoutthe disclosure, the following terms, unless otherwise indicated, shallbe understood to have the following meanings:

“Adaptive routing” refers to a change in the path to be followed by awork unit as a result of conditions encountered during a stage or stagesof processing. Conditions could include results of previous processingsteps, equipment out of order, processing priorities, or other factors.The path is the sequence of steps called for to process a work unit. Forexample, the path is the sequence of steps called for in the assaydefinition, which may be independent of specific processing equipment.System processing is typically performed at workstations, so adaptiverouting allows alternative workstations to be substituted bycomputerized instruction.

“Daughter plate” refers to a plate containing a portion of the liquid ofthe wells of a master plate of the same or different well density. Amaster plate refers to a plate with wells containing a stock solution.

“Modulation” refers to the capacity to either enhance or inhibit afunctional property of biological activity or process (e.g., enzymeactivity or receptor binding); such enhancement or inhibition may becontingent on the occurrence of a specific event, such as activation ofa signal transduction pathway, and/or may manifest only in particularcell types.

The term “modulator” refers to a chemical compound (naturally occurringor non-naturally occurring), such as a biological macromolecule (e.g.,nucleic acid, protein, non-peptide, or organic molecule), or an extractmade from biological materials such as bacteria, plants, fungi, oranimal (particularly mammalian) cells or tissues. Modulators areevaluated for potential activity as inhibitors or activators (directlyor indirectly) of a biological process or processes (e.g., agonist,partial antagonist, partial agonist, antagonist, antineoplastic agents,cytotoxic agents, inhibitors of neoplastic transformation or cellproliferation, cell proliferation-promoting agents, and the like) byinclusion in screening assays described herein. The activity of amodulator may be known, unknown or partially known.

“Naturally-occurring” as used herein, as applied to an object, refers tothe fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

“Operably linked” refers to a juxtaposition wherein the components sodescribed are in a relationship permitting them to function in theirintended manner. For instance, two components are mechanically linked bya conveyor means.

“Parallel processing” refers to the routing of material flow tofacilitate the simultaneous handling of multiple work units at or withinmultiple workstations. Parallel processing between workstations isaccomplished by maintaining individual work queues within a transportsystem for each workstation, and allowing for many liquid handlingoperations to be performed simultaneously. For example, work units canbe delivered in parallel to each of the workstations disposed on atransport system, while other units are queuing for subsequent operationat those workstations. Transfers from the workstations are also to beaccomplished in this manner. Within workstations, many parallelinstruments can perform work on a number of units simultaneously. Forinstance, four parallel aspirate/dispense devices can simultaneouslyoperate on four plates in a workstation. When the term parallelprocessing is used, unless explicitly stated, it does not preclude othertypes of processing, such as serial processing.

“Polynucleotide” refers to a polymeric form of nucleotides of at least10 bases in length, either ribonucleotides or deoxynucleotides or amodified form of either type of nucleotide. The term includes single anddouble stranded forms of DNA.

“Chemical plate” refers to a plate containing chemcials, such as amaster plate with stock solutions or a daughter plate with stocksolutions or dilutions thereof. A well of a chemical plate will usuallyhave only one chemical in solution.

“Sample plate” refers to a plate containing a sample to be processed,such as a sample for testing or synthesis. Sample plates are usuallyused in a reaction module, to permit a chemical reaction, or detectionof a physical property of the sample.

“Test chemical” refers to a chemical to be tested by one or morescreening devices or method(s) of the invention as a putative modulator.

“Fluorescent label” refers to incorporation of a detectable marker,e.g., by incorporation of a fluorescent moiety to a chemical entity thatbinds to a target or attachment to a polypeptide of biotinyl moietiesthat can be detected by avidin (e.g., streptavidin containing afluorescent label or enzymatic activity that can be detected byfluorescence detection methods). Various methods of labelingpolypeptides and glycoproteins are known in the art and may be used.Examples of labels for polypeptides include, but are not limited to dyes(e.g., FITC and rhodamine), intrinsically fluorescent proteins, andlanthanide phosphors. In some embodiments, labels are attached by spacerarms of various lengths to reduce potential steric hindrance.

“Reporter gene” refers to a nucleotide sequence encoding a protein thatis readily detectable either by its presence or activity, including, butnot limited to, luciferase, green fluorescent protein, chloramphenicolacetyl transferase, p-galactosidase, secreted placental alkalinephosphatase, β-lactamase, human growth hormone, and other secretedenzyme reporters. Generally, reporter genes encode a polypeptide nototherwise produced by the host cell which is detectable by analysis ofthe cell(s), e.g., by the direct fluorometric, radioisotopic orspectrophotometric analysis of the cell(s) and preferably without theneed to remove the cells for signal analysis of a well. Preferably, thegene encodes an enzyme which produces a change in fluorometricproperties of the host cell which is detectable by qualitative,quantitative or semi-quantitative function of transcriptionalactivation. Exemplary enzymes include esterases, phosphatases, proteases(tissue plasminogen activator or urokinase) and other enzymes whosefunction can be detected by appropriate chromogenic or fluorogenicsubstrates known to those skilled in the art. Proteins, particularlyenzymes, of reporter genes can also be used as probes in biochemicalassays, for instance after proper conjugation to either the target or achemical entity that binds to the target.

“Pharmaceutical agent or drug” refers to a chemical compound orcomposition capable of inducing a desired therapeutic effect whenproperly administered to a patient.

Other chemistry terms herein are used according to conventional usage inthe art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(ed. Parker, S., 1985, McGraw-Hill, San Francisco, incorporated hereinby reference).

Introduction

The present invention is directed to systems and methods that utilizeautomated and integratable workstations for identifying chemicals havinguseful activity. The present invention is also directed to chemicalentities and information (e.g., chemical or biological activities ofchemicals) generated or discovered by operation of workstations of thepresent invention.

The present invention typically includes automated workstations that areprogrammably controlled to minimize processing times at each workstationand can be integrated to minimize the processing time of the liquidsamples from the start to finish of the process. Typically, a system ofthe present invention would include most or all of components used inprocessing liquid samples to identify a useful chemical, starting with alarge store of different reagents (usually liquid) through the laterstage processing steps, such as chemical reactions, or detection of ananalyte or measurement of a physical property of a sample, as well as acomponent to collect information resulting from such a process. Such asystem, as shown in FIG. 1, usually includes the following components:

a) a storage and retrieval module for storing and retrieving very largenumbers (at least about 100,000) of different reagents in containers,

b) a sample distribution module to handle (e.g., aspirate samples fromcontainers and dispense samples into sample containers) small volumes ofliquids at a high rate of speed,

c) a sample transporter to transport reagents from a selected componentto another at a compatible throughput rate,

d) a reaction module (e.g., a reagent dispenser or a detector) forchemical reactions or physical measurements at high throughput rates,and

e) a data processing and integration module that can control moduleoperation.

If desired, each separate module is integrated and programmablycontrolled to facilitate the rapid processing of liquid samples, as wellas being operably linked to facilitate the rapid processing of liquidsamples.

As a non-limiting introduction to the breadth of the invention, theinvention includes several general and useful aspects, including:

1) ensembles of integrated components to form a system (e.g., a systemthat includes a storage and retrieval module, sample distributionmodule, sample transporter, reaction module and data processing andintegration module),

2) individual components (e.g., storage and retrieval module, sampledistribution module, sample transporter, reaction module or dataprocessing and integration module),

3) methods of identifying useful chemicals,

4) chemicals discovered by the operation of such components or systems,

5) methods and compositions for modulating biological processes with achemical having modulating activity discovered by the operation of suchcomponents or systems, and

6) computer program products, computational methods and storage devicesrelated to either the operation of, or information generated by,components or systems of the invention.

These aspects of the invention, as well as others described herein, canbe achieved by using the devices, compositions and methods describedherein. To gain a full appreciation of the scope of the invention, itwill be further recognized that various aspects of the invention can becombined to make desirable embodiments of the invention. For example,the invention includes a storage and retrieval module programmablyintegrated with a computer device to a sample transporter and a sampledistribution module, and operably linked to the sample transporter andthe sample distribution module. Such combinations result in particularlyuseful and robust embodiments of the invention.

FIG. 1 shows one embodiment of a liquid processing system that comprisesa storage and retrieval module, sample distribution module, sampletransporter, reaction module and data processing and integration module.As described in further detail herein, the data processing andintegration module can integrate the various modules and provide forcomputer control of the modules and sample transporter, as well asrouting (e.g., adaptive routing), and parallel processing of work to beperformed by each workstation. Information transfer can occur from thedata processing and integration module to a workstation and from aworkstation to a data processing and integration module. Materialtransfer, in this case plates, can occur between modules and isfacilitated by operably linking the modules together with the sampletransporter. Because the invention permits tremendous flexibility forintegrating liquid processing workstation, many embodiments of theinvention are contemplated, such as in FIG. 2, which shows a liquidprocessing system comprised of an additional workstation that could beused for identifying useful chemicals, as described in more detail.

The present invention is described more fully herein first as a systemand then on a component-by-component basis. Specific examples of systemsand methods suited to particular applications, chemicals discovered bythe present invention and methods relating to modulating biologicalprocesses with such chemicals are then described. The Examples arepreceded by a description of computer pro gram products, computationalmethods and storage devices related to either the operation of, orinformation generated by, components or systems of the invention.

System for Rapidly Processing Liquid Samples

The invention provides for a system for rapidly processing liquidsamples, comprising workstations that are programmably integrated andoperably linked to facilitate processing of liquid samples and tominimize processing time. The system can include a storage and retrievalmodule that can house a plurality of chemicals in solution inaddressable chemical wells. A chemical well retriever is disposed tostore and retrieve addressable chemical wells that can be stored in thestorage and retrieval module. The storage and retrieval module can becomputer-controlled to offer programmable selection and retrieval of theaddressable chemical wells. The storage and retrieval module canoptionally include an addressable well sorter that acts as a buffer fortemporary addressable well storage.

A sample transporter is operably linked to the storage and retrievalmodule to transport the selected addressable chemical wells to a sampledistribution module. The sample transporter is optionally programmablycontrolled to direct transport of the selected addressable chemicalwells to the sample distribution module.

The sample distribution module comprises a liquid handler to aspirate ordispense solutions from selected addressable chemical wells. The sampledistribution module can provide for a programmable selection of andaspiration from the selected addressable chemical wells and programmabledispensation into addressable sample wells. The addressable sample wellscan contain other chemicals required for processing, synthesis oranalyte detection.

To accomplish further processing, a reaction module is providedcomprising either a reagent dispenser to dispense reagents (e.g.chemicals) into the addressable sample wells for a reaction or adetector to detect chemical reactions in the addressable sample wells.

The modules can be separately controlled or programmably controlled andintegrated using a data processing and integration module. The dataprocessing and integration module permits orchestrated processing todeliver addressable chemical wells or addressable sample wells toworkstations so as to reduce processing time and permit, if so desired,parallel processing of addressable chemical wells or addressable samplewells. Typically, the storage and retrieval module, sample distributionmodule, sample transporter, reaction module and data processing andintegration module are operably linked to facilitate rapid processing ofthe addressable sample wells or the addressable chemical wells.

The invention's system permits high-throughout processing of liquidsamples, and storage and retrieval from vast stores of chemicals storedin solution. Typically, the storage and retrieval module has storagelocations for at least 200,000 addressable chemical wells. To facilitatestorage and retrieval, such wells are often organized or integrated intoa plurality of addressable chemical plates that can hold multipleaddressable chemical wells (or any other type of addressable well). Thestorage and retrieval module is operably linked to at least one platebuffer that loads and unloads the addressable chemical plates in apredetermined order that is either dependent or independent of the orderof selection of the addressable chemical plates. This feature has theadvantage of retrieving chemicals plates from the buffer in a mannerthat minimizes processing time at workstations downstream of the platebuffer, while minimizing retrieval time from the storage and retrievalmodule. The chemical well retriever is adapted to retrieve and replaceselected addressable chemical plates and can be controlled by a programto reduce retrieval and replacement time of the chemical well retriever.

The storage and retrieval module can also provide storage locations foraddressable sample wells. Usually addressable sample wells will beorganized or integrated into a plurality of sample plates. It isdesirable to design each sample plate with a standard footprint andpreferably to match the sample plate footprint with the footprint of thechemical plate. The chemical well retriever can be adapted to store andretrieve selected addressable sample plates, as well as being adapted toretrieve addressable sample plates of a different thickness than theaddressable chemical plates. Typically, a storage and retrieval modulecan house at least 20,000 addressable sample wells. This feature offersthe advantage of reducing the types of retrievers required for storingand retrieving plates while permitting storage and retrieval of plateshaving wells of a different depth (hence the difference in platethickness) and well volume.

According to the invention, it will be advantageous to reduce the volumeof the chemical or sample processed. Liquid sample processing timesbenefit from volume reduction because liquid dispensing times arereduced, liquid aspiration times are reduced, diffusion times afteraddition of a reagent or sample are decreased and temperature control ofa smaller volume is more uniform and consumable costs are greatlyreduced. To reduce reagent (or chemical) volumes and permit dilutioninto smaller samples, the sample distribution module can include aliquid handler that comprises a plurality of nanoliter dispensers thatcan individually dispense a predetermined volume of less thanapproximately 2,000 nanoliters of liquid from a predetermined selectionof addressable chemical wells into a predetermined selection ofaddressable sample wells. Preferably, a liquid handler comprises aplurality of nanoliter dispensers that can individually dispense apredetermined volume of liquid from a predetermined selection of saidaddressable chemical wells into a predetermined selection of saidaddressable sample wells. The nanoliter dispensers will typically have acenter- to-center distance between each nanoliter dispenser of less than9.0 mm. This feature permits liquid handling in conjunction with avariety of plate formats, as described herein. Different types ofnanoliter and picoliter dispensers can be used as described herein andknown in the art, as well as such dispensers developed in the future.

In many processing applications it is desirable to include a reactionmodule. The reaction module can be a reagent dispenser to permitchemical reactions, or addition of assay components (e.g., chemicals orcells), or a detector to detect chemical reactions, or changes in thephysical properties of a sample in an addressable sample well. Asdescribed in greater detail herein, optical detection methods are oftencost effective and permit high-throughput of samples. Preferably, thedetector is optically disposed to launch light into selected addressablesample wells as part of an optical based detection module.

To facilitate the routing of addressable chemical or sample wells, thedata processing and integration module can be programmed to route workto various workstations to minimize transport time or processing timeassociated with traveling to multiple workstations. The data processingand integration module can control the retrieval of selected addressablechemical wells, aspiration of selected chemicals from addressablechemical wells, transport along predefined routes, transport ofaddressable sample wells to the sample distribution module, dispensinginto selected sample wells, routing the sample wells to a detector andcollecting and storing data output from the detector. The dataprocessing and integration module can also include an adaptiveprocessing program to route the addressable sample wells and addressablechemical wells to workstations.

According to the present invention, ultra high-throughputs of liquidsamples can be achieved using an integrated and operably linked system.Typically, an integrated system will integrate the modules of the system(e.g., the storage and retrieval module, sample distribution module, anddata processing and integration module) and operably link such modulestogether with a sample transporter to process addressable sample wellsat a rate of about 100,000 per day. Such ultra high-throughput rates areeasier to achieve when the sample distribution module, sampletransporter and reaction module are adapted to process miniaturizedaddressable sample wells having sample volumes of less than about 5 to10 microliters in a well.

Storage and Retrieval Module

The invention provides for a storage and retrieval module that canindividually store a plurality of chemicals in solution. Typically, eachchemical(s) is individually stored in a well. Typically, the wells canbe addressed (addressable wells) by a computer, e.g., a means toinstruct the storage or retrieval of a particular well. The computerinstructions can be for an isolated single well(s) or for an array ofwells on a plate. Plates and wells can be identified with anidentification system such as a bar code. Well storage and retrieval canoffer programmable selection of addressable chemical wells. Oneembodiment of the storage and retrieval module is shown in FIG. 5 thatis operably linked to a sample transporter.

The storage and retrieval module also comprises a chemical wellretriever that is computer-controlled to store and retrieve addressablewells in the storage and retrieval module . The chemical well retrievertypically retrieves or stores addressable wells in a predeterminedlocation with specified X and Z coordinates. Preferably, the chemicalwell retriever comprises a movable buffer that permits the chemical wellretriever to load and unload multiple addressable wells without havingto re-position the chemical well retriever. This allows the chemicalwell retriever to reduce its travel time and distance in the storage andretrieval module by carrying addressable wells (e.g., multiple arrays ofwells as plates) during its travel in the storage and retrieval modulerather than individually storing and retrieving each plate from astorage location and back to an unload location. Often the chemical wellretriever will be able to handle plates with lids. Preferably, thechemical well retriever can handle any type of plate with apredetermined footprint. Preferably, the chemical well retriever willnot use a robotic hand that grips the plate from the plate's side.Instead, it preferably retrieves a plate from the bottom, for example,with a platen. The chemical well retriever is usually not used as asample transporter, as described herein. By providing a universal platehandler, the chemical well retriever provides for a flexible storage andretrieval system. One such example of a universal plate handler isdescribed herein in the section describing screening applications of theinvention.

Typically, the storage and retrieval module will handle plates with astandard footprint, either a predetermined standard or an industrystandard. “Standard footprint” refers to the X,Y dimensions of a plate(length and width). Changes in storage and retrieval density can beachieved by varying the well-to-well distance. Preferably, the standardfootprint is about 12.8 cm by about 8.5 cm. The plates can be housed inplate hotels that have dimensions that allow the plate to be stored andwill provide sufficient space to permit a chemical well retriever toaccess a particular plate. Preferably, the chemical well retrieverengages the plate from a bottom portion of the plate to reduce thedimensions of the plate hotel and eliminate dependency on a particularplate geometry. A chemical well retriever may be used as describedherein for screening applications, as well as other devices capable ofhandling addressable wells as known in the art or developed in thefuture. The storage and retrieval module can have storage locations forat least about 25,000 to 50,000 addressable wells, about 500,000addressable wells, or about 1,000,000 addressable wells, or about10,000,000 addressable wells. The storage retrieval module can alsoinclude hotels on vertical movable members to access and sort thehotels.

In most embodiments it will be advantageous to integrate and operablylink the storage and retrieval module with at least one otherworkstation, usually a sample transporter. The integration can beaccomplished with a computer and associated control programs to instructthe chemical well retriever. For implementation with a liquid processingsystem, a data processing and integration module type device may be usedas described herein, as well as other computing devices capable ofintegrating instrumentation as known in the art or developed in thefuture. Alternatively, the storage and retrieval module may be usedwithout directly integrating it with another workstation by trackingaddressable wells in groups and either mechanically or manuallytransporting the addressable wells to another work station where theaddressable wells are identified. While this approach is feasible,especially for lower throughputs, it is not desirable for higherthroughputs as it lacks direct integration that can lead to fasterthroughput times and can rely on manual operations that are morefrequently subject to error, especially when processing large numbers ofsamples. Preferably, the storage and retrieval module can be integratedwith other workstations and operate in a mode with minimal orsubstantially no manual operations related to transferring addressablewells to other work stations.

In some embodiments, especially storage and retrieval modules fortemperature or atmosphere sensitive chemicals, it will be desirable toinclude an environmental control unit to modulate the environment of thestorage and retrieval module (e.g., temperature, inert gases, andhumidity of the storage and retrieval module). This can be accomplishedby housing the storage and retrieval module, including the addressablewell retriever, in an insulated casing with a suitable control unit(s).Preferably, a suitable control unit can maintain temperatures of eitherabout 20 to 25 degrees Celsius, 0 to 5 degrees Celsius or−20 to −25degrees Celsius. If desired, a dehumidifier can be included to removemoisture when either the chemicals or solvent have a tendency to absorbwater. The storage and retrieval module is usually designed forsemi-permanent storage (at least about 30 days to 120 days) and canaccommodate retesting of chemicals.

FIG. 3 shows one embodiment of a storage and retrieval module withhotels 110 and racks 120 for plates. A chemical well retriever 130stores and retrieves addressable wells in plates from racks in hotelsand is disposed on horizontal and vertical track 140 that can positionthe retriever. A chemical well retriever arm 150 that engages platesfrom the bottom to place plates on, and remove plates from, a rack. Aremovable store 160 that can be manually removed is also shown. Thestorage and retrieval module can be operably linked to a sampletransporter at an ingress/egress junction 170.

Sample Distribution Module

The invention provides for a sample distribution module that candispense or aspirate large numbers of solutions, usually small volumesolutions. When the sample distribution module is integrated with astorage and retrieval module, it will be advantageous for the sampledistribution module to both aspirate and dispense solutions with aliquid handler. In many instances, the sample distribution module willhold large numbers of different stock solutions of chemicals dissolvedin aqueous or non-aqueous solvents (e.g., water or dimethylsulfoxide(DMSO)) in addressable chemical wells. To facilitate the rapid transferof these stock solutions, it is desirable for the sample distributionmodule to aspirate a stock solution from an addressable well anddispense all or a portion of that solution into an addressable samplewell or another addressable well. This sequence of events can beprogammably controlled to ensure that the stock solution is aspiratedfrom a pre-selected addressable chemical well and is dispensed into apre-selected addressable sample well. This type of sample distributionmodule and process is useful for generating daughter plates from masterplates or for transferring and diluting a chemical solution from achemical plate to a sample plate. Typically, a sample transporter can beused to mechanically link the sample distribution module to a storageand retrieval module for the preparation of daughter plates. The sampledistribution module can also be integrated to other components, forinstance a conveyor transport system can link a sample distributionmodule to a reaction module.

It is particularly advantageous for the sample distribution module tohandle plates of different well densities. This is usually accomplishedby a sample distribution module that can recognize plates with astandard footprint. Plates having different well densities but similarstandard footprints can then be processed. When handling plates ofdifferent densities, it is desirable to track the plate density with adatabase linked to a plate bar code (or some other plate identificationsystem, e.g., radio frequency) and to provide a sample distributionmodule that can register the bar code. When used with a data processingand integration module controller, the bar code can easily reference aplurality of plate and well information from the data store, such thatno encoded data is necessary on the bar code itself. The sampledistribution module can then be properly instructed to aspirate ordispense in a manner that corresponds to the well density of the plate.This permits aspiration at one well density and dispensation at a secondwell density. Thus, compression of low density plates can occur by thetransfer of liquids to a higher density plate and expansion of highdensity plates can occur by transfer of liquids to a lower densityplate. This feature advantageously allows a sample distribution moduleto functionally interface with other workstations that may individuallyutilize plates of different well density.

For example, traditional 96-well plates can be used to store chemicalsolutions in master plates in a storage and retrieval module. The sampledistribution module aspirates a predetermined volume of chemicalsolution from all the addressable chemical wells of a master plate. Thesample distribution module then dispenses a predetermined volume ofchemical solution into a pre-selected portion of the addressable wellsof a 384 daughter plate (i.e. compression). This process can be repeatedto construct replicate arrays on the same or different daughter plate.

Aspiration or dispensation into plates of different densities can beaccomplished by automated orthogonal positioning of a plate. Typically,the plates are securely disposed on an orthogonal positioner that movesthe wells of a plate with a first density in an X,Y position withrespect to the X,Y position of the liquid handler. Usually, the liquidhandler will have an array of aspiration and/or dispensation heads, orboth. Many aspiration/dispensation heads can operate simultaneously. Theorthogonal positioner will align each well with the appropriatedispensing head. Preferably, a predetermined location (e.g., center) ofa pre-selected addressable well will be aligned with the center of adispensing head's fluid trajectory. Other alignments can be used, suchas those described in the examples. With a head substantially smallerthan a well diameter, orthogonal positioning permits aspiration ordispensation into plates of different densities and well diameters.

An orthogonal positioner can typically match an array of dispensingheads with an array of wells in X,Y using a mechanical means to move thewells into position or the liquid handler (e.g., dispensing heads) intoposition. Preferably, arrays of wells on a plate are moved rather thanthe liquid handler. This design often improves reliability, since platesare usually not as heavy or cumbersome as liquid handlers, which resultsin less mechanical stress on the orthogonal positioner and greatermovement precision. It also promotes faster liquid processing timesbecause the relatively lighter and smaller plates can be moved morequickly and precisely than a large component. The mechanical means canbe a first computer-controlled servo motor that drives a base disposedon a X track and a second computer-controlled servo motor that drives aY track disposed on the X track. The base can securely dispose a plateand either a feedback mechanism or an accurate Cartesian mapping system,or both that can be used to properly align wells with heads. Other suchdevices, as described herein, known in the art or developed in thefuture to accomplish such tasks can be used. Usually, such devices willhave an X,Y location accuracy and precision of at least ±0.3 mm in X and±0.3 mm in Y, preferably of at least ±0.09 mm in X and ±0.09 mm in Y,and more preferably of at least ±0.01 mm in X and ±0.01 mm in Y. It isdesirable that such devices comprise detectors to identify the wells orplates being orthogonally positioned. Such, positioners forpredetermined X, Y coordinates, can be made using lead screws having anaccurate and fine pitch with stepper motors (e.g., Compumotor Stagesfrom Parker, Rohnert Park, Calif., USA). Such motors can becomputer-controlled with the appropriate electrical inputs to thestepper motor. Orthogonal positioners can be used with other componentsof the invention, such as the reagent dispenser or detector to positionsample plates. Alternatively, the liquid handler can be disposed on aZ-positioner, having an X,Y positioner for the liquid handler in orderto enable precise X,Y and Z positioning of the liquid handler (e.g.,Linear Drives of United Kingdom).

A reference point or points (e.g., fiducials) can be included in the setup to ensure that a desired addressable well is properly matched with adesired addressable head. For instance, either the plate, the orthogonalpositioner or the liquid handler can include a reference point(s) toguide the X,Y alignment of a plate, and its addressable wells, withrespect to the liquid handler. For example, the liquid handler has adetector that corresponds in X,Y to each corner of a plate. The platehas orifices (or marks) that correspond in X,Y to the liquid handler'sposition detectors. The plate's orifices allow light to pass or reflectfrom a computer-controlled identification light source located on theorthogonal positioner in the corresponding X,Y position. Opticallocators known in the art can also be used in some embodiments (PCTpatent application WO91/17445 (Kureshy)). Detection of light by theliquid handler emitted by the orthogonal positioner verifies thealignment of the plates. Once plate alignment is verified, aspiration ordispensation can be triggered to begin. Stepper motors can be controlledfor some applications as described in U.S. Pat. No. 5,206,568(Bjornson).

The liquid handler will also typically be disposed on a Z-dimensionalpositioner to permit adjustments in liquid transfer height. This featureallows for a large range of plate heights and aspirate and dispensetips, if desired, to be used in the sample distribution module. It alsopermits the dispense distance between a well surface, or liquid surfacein a well, and a liquid handler to be adjusted to minimize the affectsof static electricity, gravity, air currents and to improve the X,Yprecision of dispensation in applications where dispensation of a liquidto a particular location in a well is desired. Alternatively, plates canbe positioned on a Z-dimensional positioner to permit adjustments inliquid transfer height. Static neutralizing devices can also be used tominimize static electricity. Generally, the liquid transfer height willbe less than about 2 cm. Preferably, small volumes will be dispensed ata liquid transfer height of less than about 10 mm, and more preferablyless than about 2 mm. Occasionally, it may be desirable to contact thetips with a solution in a controllable fashion, as described herein orknown in the art

The sample distribution module can be structured to minimizecontamination. The liquid handler can be constructed to offer minimumtip exposure to liquids using a sensor (e.g., acoustic, and refractiveindex). For instance, probe contact with a liquid surface can be reducedby providing a liquid sensor on the dispensing tip, such as aconductivity or capacitance sensor, that fonns a feedback system tocontrol the entrance of a tip into a liquid. Carryover from one plate toanother plate can be kept to acceptable level with a blow-out of the tipand minimizing tip penetration into a liquid with a sensor. Preferably,a sample distribution module will include a means for volume control,and washing the liquid handler. Alternatively, the data processing andintegration module can calculate the remaining levels in the wells basedon usage and predicted evaporation, in order to deploy the tips tosuitable measured distance and can be adjusted for plated of differentheights.

The sample distribution module will often include a plate buffer (e.g.,a stacker). The buffer acts as a temporary storage depot for addressablewells or plates. Preferably, plate retrieval from a plate buffer will bepredetermined. Plate retrieval can be either dependent or independent ofthe order of selection and can be computer-controlled. Preferably, thedata processing and integration module will include a routine to reducestorage and retrieval time, as described herein. It is also desirable toprovide for a routine to reduce transport time of any well retrieverused in astorage and retrieval module. By allowing a plate buffer toacquire addressable wells as they are retrieved by an storage andretrieval module, the transport routine of the storage and retrievalmodule can be designed to minimize retrieval time rather than toretrieve addressable wells in a sequential order.

A stacker can be used as a plate buffer. Typically, a plate stacker willup/down stack plates of a standard footprint and with differentdensities (e.g., deep well (e.g., 5 cm) or shallow well microplates of96 (e.g., 1 cm), 384,864, and 3,456 (e.g., 1 to 3 mm) well format orgreater (e.g., 6,912 or 13,024)). A computer control system will trackstacker contents. The stacker can optionally include a delidder toremove lids on lidded plates.

The operation of the sample distribution module will usually be highlyflexible to satisfy the needs of different liquid processingapplications. Predefined operations can be made available for selectionby an end user, or end users may create an entirely new method.Operations can be performed on a wide variety of plates and batch sizesof plates can vary. Sample plates and chemical plates may be selectedwith a different format from distribution plates (e.g., daughterplates). The sample distribution module will usually have the ability topool samples. The report of success, failure and errors can be sent backto a control processor or to a display for the end user. The sampledistribution module will typically provide for a stand alone mode andcan be preferably integrated with a data processing and integrationmodule.

In one embodiment, the liquid handler can comprise a plurality ofnanoliter dispensers that can individually dispense a predeterminedvolume. Typically, dispensers are arranged in two-dimension array tohandle plates of different well densities (e.g., 96, 384, 864 and3,456).

Usually, the dispensed volume will be less than approximately 2,000nanoliters of liquid that has been aspirated from a predeterminedselection of addressable chemical wells and dispensed into apredetermined selection of addressable sample wells. Preferably,nanoliter dispensers can dispense less than approximately 500nanoliters, more preferably less than approximately 100 nanoliters, andmost preferably less than approximately 25 nanoliters. Dispensing below25 nanoliters can be accomplished by dispensers described herein.Preferred, minimal volumes dispensed are 5 nanoliters, 500 picoliters,100 picoliters, 10 picoliters. It is understood that dispensers capableof dispensing such minimal volumes are also capable of dispensinggreater volumes. The maximal volume dispensed will be largely dependenton the dispense time, reservoir size, tip diameter and dispenser type.Maximum volumes dispensed are about 10.0 microliters, 1.0 microliters,and 200 nanoliters. Preferably, such liquid handlers will be capable ofboth dispensing and aspirating. Usually, a nanoliter dispenser (orsmaller volume dispenser) comprises a fluid channel to aspirate liquidfrom a predetermined selection of addressable wells (e.g., chemicalwells). Liquid handlers are further described herein, and for somevolumes, typically in the microliter range, suitable liquid dispensersknown in the art or developed in the future can be used. It will beparticularly useful to use liquid handlers capable of handling about 1to 20 microliter volumes when it is desired to make daughter plates frommaster plates. Preferably, in such instances a liquid handler has adispensing nozzle that is adapted for dispensing small volumes and cansecure a tip having a fluid reservoir.

In one embodiment nanoliter dispensers comprise solenoid valves fluidlyconnected to a reservoir for liquid from an addressable chemical well.The fluid reservoir can be a region of a dispenser tip that can holdfluid aspirated by the nanoliter dispenser. Usually, a tip reservoirwill hold at least about 100 times the minimal dispensation volume toabout 10,000 times the dispensation volume and more preferably about250,000 times the dispensation volume. The solenoid valves control apositive hydraulic pressure in the reservoir and allow the release ofliquid when actuated. A positive pressure for dispensation can begenerated by a hydraulic or pneumatic means, e.g., a piston driven by amotor or gas bottle. A negative pressure for aspiration can be createdby a vacuum means (e.g., withdrawal of a piston by a motor). For greaterdispensing control, two solenoid valves or more can be used where thevalves are in series and fluid communication.

In another embodiment, nanoliter dispensers comprise an electricallysensitive volume displacement unit in fluid communication to a fluidreservoir. Typically, the fluid reservoir holds liquid aspirated from anaddressable chemical well. Electrically sensitive volume displacementunits are comprised of materials that respond to an electrical currentby changing volume. Typically, such materials can be piezo materialssuitably configured to respond to an electric current. The electricallysensitive volume displacement unit is in vibrational communication witha dispensing nozzle so that vibration ejects a predetermined volume fromthe nozzle. Preferably, piezo materials are used in dispensers forvolumes less than about 10 to 1 nanoliter, and are capable of dispensingminimal volumes of 500 to 1 picoliter. Piezo dispensers can be obtainedfrom Packard Instrument Company, Conn. USA (e.g., an accessory for theMultiProbe 104). Such devices can also be used in other liquid handlingcomponents described herein depending on the application. Such smalldispensation volumes permit greater dilution, conserve and reduce liquidhandling times.

In some embodiments, the liquid handler can accommodate bulkdispensation (e.g., for washing). By connecting a bulk dispensationmeans to the liquid handler, a large volume of a particular solution tobe dispensed many times. Such bulk dispensation means are known in theart and can be developed in the future.

In most embodiments, it will be advantageous to integrate and operablylink the sample distribution module with at least one other workstation,usually a sample transporter. The integration can be accomplished with acomputer and associated control programs to instruct the liquid handler.For implementation with a liquid processing system, a data processingand integration module type device may be used as described herein, aswell as other computing devices capable of integrating instrumentationas known in the art or developed in the future. Alternatively, thereaction module may be used without directly integrating to anotherworkstation by tracking addressable wells in groups and eithermechanically or manually transporting addressable wells to another workstation where the addressable wells are identified. For instance, thereaction module may be directly integrated and operably linked to astorage and retrieval module and sample transporter, and indirectlylinked to a separate detector through manual operations. While thisapproach is feasible, especially for lower throughputs, it is notdesirable for higher throughputs as it lacks direct integration that canlead to faster throughput times. Manual operations also are morefrequently subject to error especially when processing large numbers ofsamples. Preferably, the reaction module can be integrated with otherworkstations and operate in a mode with minimal or substantially nomanual intervention related to transferring addressable wells to otherwork stations.

FIG. 4 shows one embodiment of a sample distribution module with aconveyor means 210 comprising a rotating band that runs the length ofthe sample distribution module platform 220 and can transport plates. Abar code reader 230 registers plates on the conveyor means. A series ofliquid handlers 240 are disposed along the conveyor means. Addressablewells in four chemical plates can be simultaneously aspirated from theliquid held in the liquid handlers, and then dispensed in additionalplates. Lids can be removed or replaced by the delidder/lidder 250.Proximal plate stacker 260 and distal plate stackers 260 can be usedtemporarily to store plates, such as chemical and sample plates, whichcan facilitate plate selection. The sample distribution module can beoperably linked to a sample transporter at an ingress/egress junction270.

Sample Transporter

The present invention provides for a sample transporter that cantransport addressable wells on plates, or units of work to workstations. A sample transporter can use series or parallel routing, aswell as combination routing that uses a combination of series andparallel processing. Such combinations can reduce transport times byoffering programmed, flexible routing to minimize transport timesbetween work stations. This also permits enhanced processing time atworkstations to reduce overall processing times. Multiple parallelpathways can also enhance flexible processing. Typically, the sampletransporter will comprise at least two parallel lanes and preferably atleast four parallel lanes. Typically, a sample transporter lane cantransport addressable wells (e.g., addressable chemical or sample wells)in both directions, bidirectional transport (e.g., north and southmovement in the same lane but at different times) by changing thetransport direction. It will, however, be desirable in some instances todedicate one or more lanes, to unidirectional transport to reducetransition times associated with changing transport direction. Each lanecan be disposed, with one or more intersections that permits transportof addressable well(s) in and out of each lane. Such intersection can beused to route addressable wells to workstations. Typically, at least oneor two workstations will be operably linked to the sample transporter,however, more workstations (e.g., 3 to 6 or more) can be operably linkedto obtain maximum benefit from flexible routing with intersections andparallel processing of complex processes.

In one embodiment, the sample transporter is a reduced friction,ortho-multilane conduit. In this embodiment, the sample transporter usesmultiple lanes to transport addressable wells straight to a destinationpoint, preferably not in a rotary fashion. Such lanes can be used tocreate processing grids comprised of intersections and highways todirect the trafficking of addressable wells. Preferably, such grids arelocated in the same plane but multi-plane grids can be used. Althoughtransport in each lane can be stopped to permit passage of a platethrough an intersection, each lane is a continuous conduit that allowsplates to flow. The plates can rest on a moving surface of the conduitor can be secured either on the conduit's surface or side, so long asthe conduit's surface allows for transport. For ease of overalloperation, the flow of the wells can be computer-controlled. In somelimited, simple applications a lane may be simply activated ordeactivated by the presence of an object as a plate without utilizing acomputer. Preferably, the conduit uses a surface material to reducefriction to minimize the force required for movement and to increase thesmoothness of transport to reduce spills, contamination, and to allowfor settling of well contents if so desired. Such materials includeTeflon™ and Delrin™. The materials can be used as rollers or moveablebases connected to a track that forms a lane. Often the reducedfriction, ortho-multilane conduit will be operably linked to a platebuffer disposed in a workstation to facilitate transport and allow forflexible routing of plates, such as chemical or sample plates or mightform a buffer or queue itself.

The transport capacity of the sample transporter should be commensuratewith the intended throughput of the system to which it is operablylinked. For instance, the rate of plate transport for standard plates istypically at least about 6 meters per minute, and preferably at leastabout 15 meters per minute. Lanes are typically about 15 to 25 cm inwidth and preferably about 1 to 5 meters based in length that may bebased on queuing requirements. The sample transporter will typicallysupport overall throughput rates of about 40,000 to 60,000 wells perday, preferably about 80,000 to 120,000 wells per day and morepreferably about 250,000 to 500,000 wells per day. Larger transportthroughputs can also be achieved between about 1 million and 10 millionaddressable wells, depending on well density. By using multiple lanes inconjunction with fast plate transport rates such throughputs can beachieved.

As an example of the design and operation of the sample transporter, thesample transporter can be configured in a continuous parallel processingdrug screening system to process samples more efficiently, particularlyliquid samples. This type of configuration can improve transport ofplates in a system with multiple work stations to more convenientlyachieve ultra high-throughput rates. A continuous parallel processingdrug screening system can provide for one or more of these features:fixed work unit, flexible routing, adaptive routing, queues, access toworkstations, and parallel or series processing. The overall design ofthe continuous parallel processing drug screening system offers flexibledelivery and presentation of work, especially for screening for usefulchemicals, chemical synthesis, analysis of environmental samples oranalysis of biological samples (e.g., medical diagnostics).

Work in the system is divided in two types of elements: 1) work that isdirected to a plurality of workstations and 2) work that is organizedinto specific “work units,” which have a common physical form (e.g.,plates). Some plates are used for storage, while other plates that aregenerally similar are used for screening. All operations are commandedby a supervisory control computer(s), such as a data processing andintegration module. The computers receive their work schedule from adata store which gives detailed instructions for workstations and foreach work unit. The structure of the data store can be of the typeprovided herein or those suitable data stores known in the art.

Workflow can be divided according to the major physical organization ofthe system. The system can comprise a chemical storage and retrievalmodule, a sample transporter, and various other workstations. FIG. 5shows an isometric view of a processing system.

Storage of work unit, such as plates, is accomplished with a racksystem. A plurality of individual storage shelves is contained in amodular hotel 301. Modular hotels are mounted on an adjustable tracksystem and attached to the store framework 302. The capacity of eachhotel is typically about 20 to 40 shelves. The hotels are arrayed in thestore in two dimensions, referred to as X and Z. Two opposing arrays ofshelves are provided, with their entries on opposite sides of a centralaisle 303.

Traveling along the ‘X’ axis in the central aisle is a robotic means tostore and retrieve work units. The robotic means can pick and place workunits in any location in the storage and retrieval module. The roboticmeans comprises an integrated buffer to optimize the X axis travel. Thiscan allow for the most efficient pick or place order via a programmedsequence of retrieval commands. The robotic means can operate on aplurality of plates without returning to the center input/outputlocation except when the integrated buffer is fully loaded. The roboticend actuator also travels in the vertical or ‘Y’ direction. A platendevice moves in the ‘Y’ axis to obtain units of work from the shelves,as described herein and can be used as a plate retrieval means as knownin the art or developed in the future.

Some sections of a storage and retrieval module can optionally containremovable capacity (e.g., hotels) 306. The removable hotels are the sameas fixed hotels, but are mounted to carts, which physically lock intoplace 305. Movable, but securable, carts are used to transfer largequantities of work into and out of the store. They can be used toreplenish the contents, or can provide blank media and collect finishedwork in a particular application, such as a drug screening applications.

At the ingress and egress location 304 in the center of the conveyor,there is a stacking or sorting means that can stack or sort work units.For instance, at the ingress, an upstacker creates a stack, which istransferred as a group to the robotic means. These units of work arethen individually placed in the shelves of the storage and retrievalmodule. At the egress position, a down stacker 308 to separate units ofwork, and a sorting means 309 reorders the work units, so that units ofwork are presented to a sample transporter in the exact order required.

The sample transporter 310 preferably connects to the storage andretrieval module near the center at the ingress and egress location 304to reduce travel time. The sample transporter can comprise a conveyor totransfer work units to workstations, and unload and offload means toassist the transfer of work units from devices, via photocells, a workunit inventory system (e.g., bar codes or other system). The sampletransporter is capable of moving a unit at a rapid speed from anyworkstation to any other random workstation. It offers flexible parallelrouting, unlike robotic means that only offer serial routing. This is adistinct advantage in drug discovery applications, and it can improvethe reliability and flexibility of rapid liquid processing.

The work unit transport can be divided into four lanes as FIG. 5. Thequeuing lane 311 prevents work unit build up by providing for a queuinglane for a workstation. The passing lane 312 permits rapid transport ofwork units to a distant workstation. Similarly, returning lanes providea passing lane 313 and a queuing lane 314.

At predetermined locations along a conveyor are lift and transfermechanisms (LAT) 315. Each LAT can move a unit from one lane to anotherunder program control. A LAT is able to transfer a work unit from theconveyor and to present it to a workstation or another conveyor. It cantransfer plates from the workstation to the conveyor in a similarmanner. Typically, LATs are belt driven conveyors that intersect with alane. The belts are located below the plane of a lane when the LAT isnot in operation. Upon activation a LAT is raised to provide contact ofthe belts with the bottom of the plate to be transferred. The belts thentransfer the plate to a predetermined location. The LAT is then loweredthereby releasing the plate to a workstation or conveyor.

Based on the result obtained at one workstation, conditions, or loading,the supervisory control system can order the transport system to moveunits to different workstations. In this manner, a busy ormalfunctioning workstation can be avoided. This adaptive routingcapability is particularly advantageous in an automated process,especially liquid processing, and can be implemented using a supervisorycontrol system for its operation, as described herein.

The supervisory control system can be implemented as a portion of thedata processing and integration module and contains computer devices forcontrol and data store for information storage and retrieval. Thesupervisory control system in the preferred embodiment includesspecialized computers for real-time control. These are typicallyprogrammable logic controllers, which are connected by network or serialinterface to a supervisor computer. The supervisor computer in thesupervisory control system interfaces with the programmable logiccontrollers to provide commands which direct the electromechanicaldevices in the sample transporter to move and divert plates. It alsoreceives information from the programmable logic controllers on thecurrent state of the sample transporter. The supervisor computerdetermines its actions based on information in the data processing andintegration module data store, and directly connected bar code readers.Each unit of work (plate) has a unique identifier which is referenced bythe data processing and integration module database. The sampletransporter contains a number of decision points, or locations where thesupervisory control system must determine the plates next routing. Areader at each decision point allows the supervisory computer to giveoptimal commands to the programmable logic controller. Some decisionpoints can omit the reader, if information is available elsewhere, i.e.stack and queue data in the supervisory control system itself.

The chemical transporter itself is preferably constructed in aintegratible sections. For example, in FIG. 5, there are three sectionsshown. Within these three sections, there are six workstations definedand five shown. The ingress/egress junction where the screening sampletransporter connects to the storage and retrieval module is also shown.Larger configurations can include additional transport sections tosupport additional workstations. Each transport section is usuallyindividually powered and controlled. Drive motors and control wiring arelocated on the bottom of each section 316.

The chemical transporter can accommodate a large number of work unitssimultaneously. Typically, the inventors have had as many as 36 platesqueued or moving on each transport section. Usually the screening sampletransporter will accommodate at least 10 work units on a movingsurface(s), preferably at least 25 work units, more preferably at least50, or at least 100 work units. Work that is waiting for processing at aworkstation can be queued on the screening sample transporter, such thata complete batch of work units is available as soon as the workstationmachine becomes available. The length of each conveyor section 310 isdirectly related to the maximum number of queued units expected at eachworkstation. In the present embodiment, the sections are approximatelynine feet in length, which allows the queuing of sixteen work units thesize of standard footprint (e.g., 8.5 cm by 12 cm) plates. Conveyorsknown in the art can be used in some embodiments (e.g. slip-torqueconveyor system by Shuttleworth, Ind., USA).

The chemical transporter is designed to reduce bottlenecks in thesimultaneous processing at the various workstations. Work units canusually be transferred on or off the screening sample transporter at arate of approximately one, every two to four seconds, at eachworkstation, although faster times of preferably one every 500 to 1,000milliseconds are contemplated. Since the transport is parallel innature, each of the six shown workstations can operate asynchronously.

The workstations shown in FIG. 5 represent typical devices that can beused in liquid processing and served by workflow instructions. Theworkstation shown in the bottom right comer is a work unit replicationdevice or sample distribution robot 317. The sample distribution robotis one form of sample distribution module as described herein. Thesample distribution robot represents a typical workflow within theworkstation. The sample distribution robot is herein operably linked tothe transport through a LAT 315 and can be integrated via a computerizedcontrol. It can include a number of operations, such as delidding, fouraspirate/dispense stations, and two stacker/destackers. The work flow inthe workstation provides for a processing of units. First in, last outprocessing has numerous advantages in the processing of units, includingthe ability to match lids to the appropriate work unit (if lids aredesired), and the ability to retain master work units when replicatedwork units are returned to the screening sample transporter.

With reference to FIG. 5, the workstation at 318 is a hit profilingrobot 318. The hit profiling robot is another form of a sampledistribution module, as described herein. The hit profiling robot adds asingle well access device for aspiration/dispense of a work unit. Alsoshown is a workstation that provides high capacity stacking 319.Additional units, such as a manual workstation, screening workstations,and other devices can also be included as part of this invention. Theirwork flow is similar to the devices already described.

A “handshaking logic” can be used between a lift and transfer mechanismand the sample transporter or a data processing and integration moduleand is described in FIG. 6A. Such logic can include all or at least fiveof the represented logic steps and more preferably at least eight logicsteps to control the timing of the lift and transport. The followingtable describes the I/O Point definitions given in FIG. 6A. These I/OPoints are defined for each lift and transfer station on the sampletransporter. Each lift and transfer station can move units of work(e.g., plates) from one lane to another, or transfer a plate to a workcenter located to either side.

TABLE 1 I/O Point in FIG. 6A Function Lane ONLT0-TP Request to placeplate on LT0 from TP lane To/Pass OFFLT0-FP Request to have plate exitfrom LT0 on the FP From/Pass lane LT0-TP Sense the presence of a plateat TP lane To/Pass LT0-ST0 Lift & Transfer Unit 0 State 1 bit LT0-UPMake the L&T 0 go up LT0-SU Sense when the LT0 unit is up LT0-F Turn L&T0 motor on in the F direction LT0-FP Sense a plate on LT0 in the FP laneFrom/Pass T601 Timer for stop delay LT0-DN Make LT0 go down LT0-SD SenseLT0 is down LT0-FPG Control for LT0 lane FP exit gate FP0-S1 Lane FPsensor From/Pass

FIG. 6B shows the electrical specifications used to integrate a workstation (work center) with a sample transporter. All of the representedpins can be used for the physical handshake connection between a workstation and a sample transporter. Preferably, at least five of therepresented pins are used and more preferably at least eight logicrepresented pins are used to integrate a work station with a sampletransporter or other module.

While the screening sample transporter provides parallel processing ofmultiple work units waiting or moving to multiple workstations, theworkstations themselves also can provide parallel operations. The sampledistribution robot shown has four parallel aspirate/dispense devices,and all can be operable on a different unit at the same time. This isone means of increasing throughput of the system. Since the screeningsample transporter can accommodate six or more workstations, and eachworkstation can simultaneously operate on four or more plates, a largenumber of parallel operations can be performed at any instant.

In addition, a sample transport means can be used to operably linkcomponents of a liquid processing system or components within a module.Such a sample transport means can include conveyor belts, articulatedrobotic arms, slide mechanisms, automated guided vehicles and the likeas known in the art, or developed in the future.

Reaction Module

Reagent Dispenser

In one embodiment, the invention provides for a reaction module that isa reagent dispenser to provide reagents (e.g. chemicals to be tested ortargets) to the addressable wells in a predetermined manner. The reagentdispenser is integrated to other workstations with the data processingand integration module and operably linked with the sample transporter.One or more reagent dispensers can be operably linked to the sampletransporter as shown in FIG. 5. Preferably, the reagent dispenser is ofthe type described in the Examples. Other reagent dispensers that arecompatible with the data processing and integration module and thesample transporter (if operable linkage to the sample transporter isdesired) can be used as known in the art or developed in the future.

FIG. 7 shows one embodiment of a reagent dispenser comprises an X,Ypositioner 910 with two stepper motors 920 to control the positioning ofa plate 930. The X,Y positioner places the plate in a predetermined X,Ycoordinate that corresponds to the array of liquid dispensers 940. Toposition the addressable wells of a high density formal plate under thelinear array, the X,Y positioner must preferably be able to locatepredetermined X,Y coordinate within about 50 to 200 microns. It istherefore desirable to integrate and control X,Y positioners of thereagent dispenser, as well as of other components, with the dataprocessing and integration module to finely control the stepper motors.Alternatively, the reagent dispenser can finely control the steppermotors with its own computer or programmable logic controllers.

For some embodiments of the invention, particularly for plates with 96,192, 384 and 864 wells per plate, dispensers are available forintegration into the system. Such dispensers are described in U.S. Pat.No. 5,525,302 (Astle), U.S. Pat. No. 5,108,703 (Pfost), U.S. Pat. No.5,226,462(Carl), and PCT patent application WO 95/31284 (Gordon).

Detector

In one embodiment the invention provides for a reaction module that is afluorescence detector to monitor fluorescence. The fluorescence detectoris integrated to other workstations with the data processing andintegration module and operably linked with the sample transporter.Preferably, the fluorescence detector is of the type described hereinand can be used for epi-fluorescence. Other fluorescence detectors thatare compatible with the data processing and integration module and thesample transporter, if operable linkage to the sample transporter isdesired, can be used as known in the art or developed in the future. Forsome embodiments of the invention, particularly for plates with 96, 192,384 and 864 wells per plate, detectors are available for integrationinto the system. Such detectors are described in U.S. Pat. No. 5,589,351(Harootunian), U.S. Pat. No. 5,355,215 (Schroeder), and PCT patentapplication WO 93/13423 (Akong). Alternatively, an entire plate may be“read” using an imager, such as a Molecular Dynamics Fluor-Imager 595.

FIG. 8 shows one embodiment of a fluorescence detector comprising an X,Ypositioner 1110 with two stepper motors (not shown) to control thepositioning of a plate 1110 (plain view) and 1120 (side view). The X,Ypositioner places the plate in a predetermined X,Y coordinate thatcorresponds to the array of optical collection assembly 1130. Toposition the addressable wells of a high density formal plate under thelinear array, the X,Y positioner must preferably be able to locatepredetermined X,Y coordinate within about 50 to 200 microns. It istherefore desirable to integrate and control X,Y positioners of thefluorescence detector with the data processing and integration module tofinely control the stepper motors. The filter wheel 1140 is part of theemission optical relay system that filters light emitted from each fiberoptic bundle 1160 that corresponds to a particular optical collectionassembly. An optical relay means 1152 optically connects the bundle to aphoton detection means 1150. A field lens 1170 is used to focusexcitation light from a light source 1192. A second filter wheel 1192 isused to filter excitation light. Light is then relayed to a field lens1190 and is then passed through a controllable aperture 1180. Light isthen finally focused onto the fiber optic bundle for emission intoaddressable wells.

The detector is preferably capable of fluorescence emission measurementsin the 400 to 800 rim range. Typically, the detector comprises a meansfor excitation of fluorescence in the 350 to 800 nm range. The detectoris often capable of many different operating modes that facilitate drugdiscovery assay requirements. These operating modes can include: singleexcitation wavelength with single emission wavelength detection, singleexcitation wavelength, dual emission wavelength detection, sequentialdual excitation wavelength with dual emission wavelength detection andratio measurement determination, sequential dual excitation wavelengthwith four emission wavelength detection and ratio measurementdetermination, homogeneous time resolved fluorescence with singleexcitation wavelength and single emission wavelength detection,homogeneous time resolved fluorescence with single excitation wavelengthand dual emission wavelength detection and ratio determinationmeasurement, homogeneous time resolved fluorescence with sequential dualexcitation wavelength and dual emission wavelength detection and ratiodetermination measurement, dual sequential excitation wavelengths andsingle emission wavelength detection with ratio determinationmeasurement, luminescence measurement at a single wavelength withluminescence measurement at dual wavelengths, luminescence measurementat dual wavelengths with a ratio determination, and time resolvedfluorescence emission (intrinsic dye properties with or without abinding event).

In the preferred embodiment, the detector comprises a light sourceassembly (e.g., Xenon) that can be switched between continuous andpulsed (1 kHz) output depending upon power supply. It includes anexcitation light aperture to control the excitation field of view forfluorescence. Fused silica fiber optic light guides can be used formaximum transmission of excitation and emission light. Guides areselected for ultra low auto-fluorescence and raman background. The lightcollection assembly is optimized to gather light from an addressablewell (e.g., coaxial fiber optic arrangement with a ball lens lightcollection assembly). The surfaces of the lens are preferably coatedwith anti-reflection coatings that reduce background signal levelsduring detection. The fiber optic guide/collection assembly is designedwith a predetermined physical spacing of guides/collection assembliesthat directs the excitation light to reduce optical cross talk tonegligible or acceptable levels at the collection site. The physicalspacing can also spatially correspond to the well density of theaddressable wells of the interrogated plate. Plates with well densitiesgreater than the density of light collection assemblies can beinterrogated with light collection assemblies spatially arranged toaccommodate X,Y positioning for higher well densities. This can beaccomplished with the use of a X,Y positioner as described herein forliquid handling apparatuses. Preferably, dual emission collection fiberoptical guides are used to enable simultaneous collection of twoemission wavelengths during detection without any movements of opticalrelay system. The detector can include an excitation filter “wheel” forrapidly changing an excitation interference filter or an emission filter“wheel” for rapidly changing an emission interference filter set. Thedetector can also include a Z positioner to change the distance betweenthe collection assemblies and the well or plate (usually the platebottom) to optimize signal collection. Typically, the fiber optic guidesand light collection assemblies are arranged in two dimensional arrays(e.g. corresponding to a 96 well plate) to allow for simultaneousdetection of all addressable wells or a matrix of a portion addressablewells. The detector can include a photon sensitive surface for measuringphoton emission, such as a CCD, photodiode, or a PMT. The detector canintensify the signal, and gate if desired, using a photon intensifier.Preferably, the detector can utilize a high quantum efficiency CCDwithout an intensifier for long detection integration. Alternatively,the detector can utilize PMT's or multi-site PMT's for photon detectionand quantitation.

The detector functions primarily in the epi-fluorescence mode where thepreferred illumination is from the bottom of the plate and the preferredcollection is also from the bottom of the plate. The detector canfunction in all of the above mentioned modes with bottom viewing of theplate. The detector usually is capable of three to four orders ofmagnitude of dynamic range in signal response from a single reading. Thedetector, in a preferred embodiment, utilizes a CCD chip for imaging anddetecting photons emitted from the assay wells.

The detector is typically capable of measuring the emission output atsimultaneous dual wavelengths for 96-assay wells at a time. The detectorcan make ratio determinations on the 96-assay wells based on the dualwavelength detection or excitation ratio measurements of 96-assay wellsby a change in the filter element in front of the Xenon Arc lamp source.The excitation ratio measurements can be measured serially at each setof 96-assay wells to be detected. The detector is capable of reading96-assay wells in less than a second. This is a dramatic improvementover “state-of-the-art” fluorescence readers. This is 30 to 200 timesfaster than current readers. The detector optical guide/collectionassembly has measured fluorescence from a sample with as little as5×10⁻¹² mole fluorescein in 1 microliter of solution and at twowavelengths simultaneously.

The ratio mode of the detector enables changes in signal levels withrespect to relative signal levels to be observed without complexcalibration. The ratio mode of the detector is tolerant of differencesin the quantities of isolated targets, cells or dye loading into cells.Hence, differences between wells can exist for the cells and dye levels,but within a single well, these differences can be normalized torelative change in the intensities. Without ratiometric detection,absolute signal levels can obscure the slight changes within each well.

The throughput of a detector in a screening system is often the slowstep in processing a large number of assay plates. The detector (ordetectors) usually must keep pace with the number of assay plates to beanalyzed. Ultimately, the rate limiting step in a screening experimentdetermines the number of assay plates which can be analyzed in a giventime period. For example, a preferred detector utilizes 96-arraytrifurcated fiber assemblies that enable one excitation wavelength (onebundle set) and two emission wavelengths (two bundle sets) per reading.In the case where excitation ratio measurements are required, thedetector can sequentially deliver the two excitation wavelengths andthen provide up to four emission wavelength results. Additional emissionwavelength readings can be obtained by sequential switching of theemission filters. In the case of a 3,456 plate, 36 separate readingswill be required to completely detect fluorescence from all 3,456addressable wells. At one second per reading, the detection time isapproximately 36 seconds not counting plate in/out and discreetmovements. If the transfer time of the plate in and out of the detectoris 24 seconds total plate reading time is one minute per plate. Thisresults in the processing of sixty 3,456 well plates per hour, or207,360 addressable wells per hour (4,976,640 addressable wells per 24hours).

The selection of different operating modes of the detector is oftenbased on the type of assay to be performed. Thus, the detector isusually designed with numerous modes of operation to provide flexibilityin detection. Each mode is selected based on its compatibility with aparticular set of fluorescent probes and reagents. The detection is thentailored to meet the assay's and the probe's requirements.

TABLE 2 Detection/ Per 96 Element Matrix Integration Positioner PausePer Plate Per Plate Time Move Time Time Total Total Total (msec) (msec)(msec) (msec) (sec) (min)  500 500 100 1100  40 0.66 1000 500 100 1600 58 0.96 3000 500 100 3600 130 2.16 5000 500 100 5600 202 3.36 10000 500 100 10600  382 6.36

Table 21 shows calculated detection times for a 96-matrix optical arraydetector The detector can have variable data acquisition time dependingon assay requirements. If a read/integration time is 1 sec per 96elements, a plate read time is under 1 minute. If a plate in/plate outoperation 1 minute, then thirty, 3,456 plates per hour can be read attwo wavelengths. Thirty such plates are equivalent to over 90,000samples per hour (assuming 10% of the plates are used for controls,etc.). For an eight hour work period, about 720,000 samples can beprocessed. The inventors have measured signals from microliter wells(assay volume about 2 microliters) in ratiometric fluorescentmeasurement mode in about 100 milliseconds.

Fluorescence Measurements

It is recognized that different types of fluorescent monitoring systemscan be used to practice the invention with fluorescent probes, such asfluorescent dyes or substrates. Preferably, systems dedicated to highthroughput screening, e.g., 96-well or greater microtiter plates, areused. Methods of performing assays on fluorescent materials are wellknown in the art and are described in, e.g., Lakowicz, J. R., Principlesof Fluorescence Spectroscopy, New York: Plenum Press (1983); Herman, B.,Resonance Energy Transfer Microscopy, in: Fluorescence Microscopy ofLiving Cells in Culture, Part B, Methods in Cell Biology, vol. 30, ed.Taylor, D. L. & Wang, Y.-L., San Diego: Academic Press (1989), pp.219-243; Turro, N. J., Modern Molecular Photochemistry, Menlo Park:Benjamin/Cummings Publishing Col, Inc. (1978), pp. 296-361 and theMolecular Probes Catalog (1997), OR, USA.

Fluorescence in a sample can be measured using a detector describedherein or known in the art for multi-well plates. In general, excitationradiation, from an excitation source having a first wavelength, passesthrough excitation optics. The excitation optics cause the excitationradiation to excite the sample. In response, fluorescent probes in thesample emit radiation which has a wavelength that is different from theexcitation wavelength. Collection optics then collect the emission fromthe sample. The device can include a temperature controller to maintainthe sample at a specific temperature while it is being scanned.According to one embodiment, a multi-axis translation stage (e.g., adedicated X,Y positioner) moves a microtiter plate holding a pluralityof samples in order to position different wells to be exposed. Themulti-axis translation stage, temperature controller, auto-focusingfeature, and electronics associated with imaging and data collection canbe managed by an appropriately programmed digital computer. The computeralso can transform the data collected during the assay into anotherformat for presentation.

Preferably, FRET (fluorescence resonance energy transfer) is used as away of monitoring probes in a sample (cellular or biochemical). Thedegree of FRET can be determined by any spectral or fluorescencelifetime characteristic of the excited construct, for example, bydetermining the intensity of the fluorescent signal from the donor, theintensity of fluorescent signal from the acceptor, the ratio of thefluorescence amplitudes near the acceptor's emission maxima to thefluorescence amplitudes near the donor's emission maximum, or theexcited state lifetime of the donor. For example, cleavage of the linkerincreases the intensity of fluorescence from the donor, decreases theintensity of fluorescence from the acceptor, decreases the ratio offluorescence amplitudes from the acceptor to that from the donor, andincreases the excited state lifetime of the donor.

Preferably, changes in signal are determined as the ratio offluorescence at two different emission wavelengths, a process referredto as “ratioing.” Differences in the absolute amount of probe (orsubstrate), cells, excitation intensity, and turbidity or otherbackground absorbances between addressable wells can affect thefluorescence signal. Therefore, the ratio of the two emissionintensities is a more robust and preferred measure of activity thanemission intensity alone.

A ratiometric fluorescent probe system can be used with the invention.For instance the reporter system described in PCT publication WO96/30540(Tsien) has significant advantages over existing reporters for geneintegration analysis, as it allows sensitive detection and isolation ofboth expressing and non-expressing single living cells. This assaysystem uses a non-toxic, non-polar fluorescent substrate which is easilyloaded and then trapped intracellularly. Cleavage of the fluorescentsubstrate by β-lactamase yields a fluorescent emission shift assubstrate is converted to product. Because the β-lactamase reporterreadout is ratiometric, it is unique among reporter gene assays in thatit controls variables such as the amount of substrate loaded intoindividual cells. The stable, easily detected, intracellular readoutsimplifies assay procedures by eliminating the need for washing steps,which facilitates screening with cells using the invention.

Data Processing and Integration Module

Because the present invention provides for unprecedented throughputrates for processing of liquid samples, it will be important in manyembodiments of the invention, especially system embodiments, tointegrate modules, direct workflow and manage information generated bysuch a system. This can be accomplished by providing a computer modulewith a processor that can integrate and programmably controlworkstations and workflow, and collect and store information from eachworkstation. A central feature applicable to most such systems is acomputer module to route the work unit, track work unit inventory and toprovide instructions to process liquid samples through the appropriateworkstations. Typically, the computer will include a relational databasethat contains information for processing a liquid sample and optionally,using the information resulting from a process to create a database ofoutput information that can then be used to support additional analysisof the data or to automatically modify the process based on the resultsobtained in a single or multiple cycles of a process.

In one embodiment, a data processing and integration module canintegrate and programmably control a storage and retrieval module, asample distribution module, and a reaction module to facilitate rapidprocessing of said addressable sample wells. Preferably, the dataprocessing and integration module can programmably route work flow tomodules by the programmably controlled adaptive routing, and optionallyprogrammably controlled parallel processing of addressable wells.

To manage information in the system, the data processing and integrationmodule comprises elements to store, manage and retrieve data, includinga data storage device and a processor. The data storage device can holda relational database, an array of physical disk drives (e.g., randomaccess disk drives), and a connection to other system components via anetwork. A data storage device can, for instance, store a relationaldatabase for environmental, diagnostic, chemical synthesis and drugdiscovery applications. For instance, one particularly useful relationaldatabase can be provided by Oracle, and the network can be a TCP/IP(transfer communication protocol) ethernet LAN (local area network).

The system can be controlled using supervisory control programs, whichare not necessarily located on the same computer as the data storagedevice. For example, in one embodiment of a system, a separatesupervisory control computer is provided for each of the Storage andRetrieval, Reagent Transport, and Reagent Distribution functions. Asupervisory control computer is a computer programmed to control aparticular subsystem using data from the data storage device andoperating on a workstation or component, such as a storage andretrieval, reaction module or sample transporter.

Within the data storage device, exists a structure for information inthe form of tables and relations. This structure is designed to meet thespecific needs of the system, wherein it must accommodate the throughputdemands of an automated system and facilitate the presentation ofinformation for analysis and visualization of results. The data storagedevice can typically process in excess of 100,000 transactions (read orwrite particular data) per day, while accurately keeping track of everychemical, biological reagent, operation, unit of work and workstationand other related activities. Integrity of the data storage device istypically maintained for simultaneous multiple users and processes.

Information in the relational database of the data storage device isused to define operations to be performed, and a complete audit trailcan be maintained of every operation on every unit of work throughoutthe system.

Storage devices suitable for use with the present invention are wellknown and are commercially available from a number of manufacturers,such as the 2 gigabyte Differential System Disk, part numberFTO-SD8-2NC, and the 10 gigabyte DLT tape drive, part number P-W-DLT,both made by Silicon Graphics, Inc., of Mountain View, Calif., orequivalents (e.g., optical discs). A preferred embodiment uses HewlettPackard 4GB Hot Swap Drives in a Netserver LX Pro configured as RAID-5.

FIG. 9 shows a data structure diagram illustrating the tables andrelations in the supervisory control system. The data structure can bedivided into function sectors, such as chemical and sample management,work flow and assay design, screening information, and subsystemcontrol.

A chemical and sample management may embody links to other computerizedsystems for data management, such as an external properties database 603that could contain chemical and physical properties. The system mayoptionally contain information related to the general lookup ofproperties or structures of chemicals. Such information is contained inthe chemical structure table 602. Chemicals themselves are representedin a chemical table 601, which contains data regarding chemical formula,scientific names, and text descriptors. All information about aparticular instance of a chemical is represented in the samples table604. A sample may be obtained from a supplier, created by a chemist, orfrom any other means defined in the source table 605. The FIG. 9 alsodescribes other relations that are most important between each table.

A chemical and sample management may also comprise a plurality ofsamples that can be grouped together in the master table 606. Theinventors have made such a collection on the order of about 10,000samples or chemicals. Larger collections of about 50,000, about 100,000and over 500,000 (e.g., about 1,000,000 to about 10,000,000) are alsocontemplated. The master table is used to define groupings of chemicals,such as would occur when the chemicals were present in a common format.

Each chemical in the master table is also represented in an aliquotstable 607. The creation of aliquots from any chemical managed by thesystem may be in any format and is not limited to multi-well plates. Achemical may be individually tracked, or become part of a masterconfiguration where it is present with a plurality of other chemicals,in which case the group, rather than a single chemical can be tracked bythe system. Each tracked entity (e.g., work unit) is recorded as anentry in format table 608. The current location of each tracked entityis stored along with its identification in the table that representsthat entity. Typical formats can include tubes and bottles of variouscapacities, arrays of wells as in the various multi-well plates, or anyother format desired. Any chemical or group of chemicals stored in aformat can be identified, e.g., by a unique bar code label.

The work flow and assay design area of the data store typically definesthe work process to be performed, and parameters necessary to controlthe automation of the system. Automation refers to both individualworkstations, and can include the transport and storage and retrievalfunctions. An external scientist interface program is used to fill invalues in the work flow and assay design tables, which can then bescheduled and executed by the operator interface program. A flowchart ofsome elements of the scientist interface is provided in FIG. 10A and oneembodiment of a scientist interface is shown in FIG. 10B.

The work flow and assay design algorithms can be implemented insupervisory control computers or with a single supervisory controlcomputer. These programs can constitute a batch process control system(meaning that desired sequences of parameters or instructions arecreated and executed in units of operation) with continuous and discreteelements (meaning that the system must manage a continuous control basedon process variables, as well as real-time events, such as interrupts).The data store structures exist to provide parameters and define theworkflow, and to receive results from these supervisory controlcomputers.

An assay can be defined in the assay table 610, composed of threeelements: selections from the chemical selection set table 611, asequence of workstations to be utilized defined in the work flow table612, and a human readable text defined in the work instructions table613. The work instructions include the steps necessary for an operatorin preparing each workstation for the assay, such as reagentpreparation, machine cleaning and setup, and any other manualoperations.

The definition of work flow can be performed by the scientist interfacesoftware. A graphical user interface is used to build an assay or otherliquid sample process on a computer screen from the various workstationsthat exist in the system. These workstations are represented in theworkstation table 614, which is itself composed of references to theequipment table 615 and the corresponding entries in the equipmentparameters table 617. Thereby, the scientist interface can expand therepresentation of a workstation to display any configurationrequirements or programs for each piece of equipment at the workstation.Different workstations will require different parameters, and screensare specially developed to enter that information in the equipmentparameters table.

Screening information portions of the data store are used to bringchemicals and work flows together and to perform a screen (oralternative an analysis or synthesis). Each screen is defined in ascreen table 620. Once screens are defined they can be scheduled forrunning by the operator interface that modifies the schedule table 621.Many screens can be defined using the same entry in the assay table 610.Based on the screen and the chemicals defined to participate in thescreen, i.e., the entries in the chemical selection set table 611 thatare defined in work flow and assay design, the operator interfaceprogram will create a list of required replicates to be created frommasters, and make entries in the replicate table 622. The replicates arealways related to a master in a particular way. Many masters can bereplicated on a replicate, but each replicate must have at least onemaster (e.g., the representation of a master plate and a daughterplate). This permits combining multiple storage plates into a singleplate of higher density.

Automated portions of the screening system, such as format copyingdevices, create replicates. The replicate table 622 is used to recordtheir parameters, i.e., the bar code label, plate type, and masterplates used. Usually it is advantageous for the replicate record itselfnot to contain information about the chemicals. For all furtheroperations on the replicate, operations will be on the entire format.For analysis purposes, the chemical information is readily available inthe master records, which are related to each replicate. The list ofreplicates for a screen is placed in a table of screen sets 623. Thescreen set is a list of the plates necessary to represent the chemicalselection set, already described in chemical selection set table 611.

The data storage device can contain items related to the automatedoperation a. process. A computer program is used to control eachworkstation in the system. An advantageous design element is to includeall information necessary to operate the workstation in the respectiveoperation table(s) 634. The operation tables given here arerepresentative and will vary for different operations that are availablein the system.

Additional tables can exist depending on the workstation to record errorcodes in the error code tables 634, history of operations in the audittrail tables 636, or raw data such as shown in the detector array table637. Subsystem results are summarized in the subsystem result table 633,which is related to the contents of the workflow table 612 defined bythe scientist interface program. For analysis, the content of thesubsystem results table 633 is used to visualize completed screens.

Previous data stores for similar applications have concentrated on thephysical representations of units of work. These systems were suited tomanual handling of units of work and often used elaborate data entrymechanisms. The present invention focuses on the automated handling ofunits of work, and typically assumes that the well quantity and densityprocessed greatly exceed what would be reasonable for a human operatorto monitor. As a result, this data store is structured for highperformance. For example, the replication of a single high density unitof work would require thousands of database transactions in otherdesigns, but can be accomplished in a single transaction against areplicate table in the present invention.

Data flow in and out of the data store is primarily through integratedinstruments, controllers, and devices. Typically, transactions e.g.,moving a plate, dispensing, aspirating, and others, are recorded in thedatabase.

Detectors, such as those found in the reaction module are instrumentswhich produce large quantities of data associated with each well. Thisdata is processed and stored in the data processing and integrationmodule. It is also desirable that the data processing and integrationmodule provide a historical record of all operations and resultsobtained for a given unit of work, or an addressable well. In thepreferred embodiment, an addressable well can be examined based on theprocessed detector results, and a hierarchical view of all previousresults and operations is obtained.

Specific Systems, Material Flow and Work Flow Related to ScreeningChemicals

One embodiment of the present invention is a liquid processing systemfor screening chemicals for useful activity, as shown in FIG. 2. Thisscreening system comprises a storage and retrieval module for storingand retrieving vast numbers of different reagents in containers, asample distribution module to handle (e.g., aspirate reagents fromcontainers and dispense reagents into other containers) small volumes ofliquids at a high rate of speed, a sample transporter to transportreagents from a selected component to another component at a compatiblethroughput rate, a hit profiling robot (HPR) to aspirate and dispenseselected solutions from addressable wells containing chemicals suspectedof having useful activity, a high capacity stacking system (HCSS) to actas a plate buffer to temporarily store plate for greater retrievalflexibility, a reaction module (e.g., a second reagent dispenser or adetector) for chemical reactions or physical measurements at highthroughput rates, and a data processing and integration module. The dataprocessing and integration module integrates each component using acomputer program, and optionally with a database comprisingpredetermined and modifiable instructions for performing the screeningprocess. The sample transporter operably integrates the systems bytransporting plates from one component to another component. Preferablythe sample transporter permits parallel processing, and optionallyadaptive routing, examples of which are described herein. The dataprocessing and integration module also preferably can integrate thesample transporter with the other components to facilitate parallelprocessing and adaptive routing of plates. Preferred components andmethods are described herein. Suitable state-of-the-art components canbe substituted, so long as such components can integrate with the dataprocessing and integration module and can be operably linked to theother components of the screening system. Other components, such asshown in FIG. 2, may be included in the system as well.

A preferred embodiment of the screening system is shown in FIG. 11.Shown in FIG. 11 is a storage and retrieval module, a random access highdensity plate presentation module, multiple reagent dispensing robotsfor dispensing reagents to be used in a substantial proportion of theplates, multiple incubators to control the temperature of chemicalevents, a fluorescent detector to detect fluorescence of each sample inan addressable well, a plate transporter to transport plates and a dataprocessing and integration module to integrate each component. This typesystem is desirable for complex screening procedures, where differentreagents may require a different reagent dispensing robot based on thetype of reagent, solvent, or screening process. One reagent dispensingrobot can be dedicated for dispensing aqueous reagents, a second fornon-aqueous solvents and a third for biological materials such as cells.Such a system can also include an incubator for controllingenvironmental conditions and humidifying assay plates to reduceevaporation. Incubators in the art can be adapted for such use, such asthose discussed in U.S. Pat. No. 5,149,654 and U.S. Pat. No. 5,525,512.Preferably, each component is serviced by a multi-lane sampletransporter to permit parallel processing.

Example of Work Unit (e.g., plate) Handling

The screening system typically operates on a work unit (unit) withstandardized processing format. The work unit sometimes embodies aparticular processing format, such as a plate. Such work units oftenvary in external dimensions, materials of construction, physical designand density (or number of wells). The screening system is preferablyconstructed using workstations to handle a standard processing formatthat allows for a flexible work unit. This approach reduces the need forcomplete uniformity of the work unit, as is required in many automatedlaboratory and chemical discovery systems. Consequently, the presentinvention's screening system can function with a mixture of many workunits without adjustment or calibration. This permits the system touniversally handle varying work units of a standardized processingformat. Usually, such systems or components will handle at least 2 workunit densities with a standard format, preferably at least 3 work unitdensities with a standard format, and more preferably at least 4 workunit densities with a standard format.

Typically, a storage and retrieval end actuator universalstacker/destacker transport system elements and the registration deviceare all devices that are designed to handle work units with standardizedprocessing format. Preferably, to enhance workflow and throughput, allthe devices that handle work units are designed to handle the samestandardized processing format. Examples of these types of devices aredescribed herein, especially in the Examples.

Material Flow Through a Screening System

The operation and liquid processing steps of a screening system can bedescribed in terms of material flow. When describing material flow, awork unit can be considered to contain one or more chemicals (orchemicals in solution) in an array. The work unit of work might be amember of the chemical library, or might be a format designed for aspecific purpose, such as a multi-well format drug screening plate. Asdescribed herein, workflow throughput can be optimized or increasedusing parallel processing, adaptive routing and computerizedinstructions from relational databases. An example of actual materialsflow through a screening system is described below.

FIG. 12 shows material flow in a screening system and pre-screeningprocessing. Master liquid storage 800, which is not usually part of thescreening system, is the long term storage of chemicals prepared incompound preparation 801. Chemicals can also be formatted into workunits in compound preparation step 801. Compound preparation involvesthe preparation of measured chemical samples, dilution in a liquid ordissolving with a solvent, and division of chemical solutions intomeasured aliquots. Usually, such chemical aliquots are reagents. In ascreening context they are often referred to as test compounds. Thechemical solutions, representing a known concentration are then groupedtogether in storage work units (e.g., addressable wells). In thepreferred embodiment, these are multi-well storage plates of high volumecapacity (e.g., 96-wells with 200 to 500 microliters per well). Astandalone sample duplication robot can aid the compound preparationprocess. Work units are then transferred directly to the storage andretrieval module using the removable cart 802 or transferred during areplenishment cycle with a removable cart 803 to the storage retrievalmodule 804. Each removable cart can contain a plurality of storage workunits (e.g., about 488). The master library work units are used toreplenish the storage retrieval module as quantities are depleted, or atime period expires. A data processing and integration module can trackthe level of solution in a storage work unit as well as the expirationdate for each work unit with a reagent or test compound.

The storage and retrieval module 804 can store a large number ofchemicals in work units. Usually, the storage and retrieval module canstore at least about 200,000 discrete chemicals on at least about 2,000thousand work units and preferably at least about 1,000,000 discretechemicals on at least about 4,000 work units and at least about10,000,000 work units. Storage volumes for such stores are describedherein (e.g., 20 to 100 microliters). Empty work units can also becontained in the store, and can be replenished with the removable cart803. The storage and retrieval module provides environmental protectionof the work units, which are individually accessible at roomtemperature. The storage and retrieval module contents can be randomlypicked and delivered to the screening transporter 805 for transport.Transport functions include presentation of work units at workstations,routing from any workstation to any other, queuing of work units atworkstations, and operably linking workstations of the screening system.

Formatting of work units (duplication or reformatting the work units thestorage and retrieval module) involves a plate replication workstation806. The equipment used in plate replication can be an sampleduplication robot. Reformatting work units involves the transfer ofchemicals from one work unit format to another, such as would berequired when a higher density work unit was needed in a screeningoperation. For example, the formatting of storage plates (often at96-wells per plate) into high density plates (e.g., as densities of atleast about 864 to 3,456-wells per plate, or higher). The platereplication workstation can efficiently create a plurality of screeningwork units from a set of storage work units. If screening work units arenot immediately needed they can be sent to the storage removal module.

As work units are directed for screening, either from the screeningtransporter 805 or from the plate replication workstation 806, they aretransferred by the screening transport 805 to the screening assay system807. Once a work unit has been used in the screening assay system, itwill usually be consumed and not replaced in the storage and retrievalmodule. In some screening applications it may be desirable to perform asecond test on the same work unit, in which case it may be desirable toroute the work unit back through the system.

The screening assay system 807 typically consists of a series of stepsdefined in an assay sequence. The scientist interface (i.e. a computerscreen or image) can be used to define each assay, which is thenrepresented in the data storage device, which is part of the dataprocessing and integration module. The material flow is modified by thesoftware definition of the assay.

The general characteristics of the screening assay system are that allwork units must follow a substantially similar material flow, in termsof time and devices used, to assure that results are comparable betweenwork units. The assay set-up with the scientist interface includes thedefinition of the material flow, timing, workstation setup requirements,reagent preparation, maximum processing time, and the work units fromthe chemical library store in the storage and retrieval module that willbe processed.

Work units 810, formatted specifically for screening by the platereplication workstation 806 are delivered by the screening transporter805. Depending on the workflow defined in the assay, they are thenconsistently processed through a series of steps. In a reagent additionstep 811, cells and a number of reagents can be dispensed into the workunit. A known agonist might be added to activate cells in a cell-basedassay. Liquid lids might be dispensed. Such liquid lids are inertmaterials, which will reduce the evaporation of liquid from a well.

The work unit is then moved to an incubation step 812. Incubation timeand environmental conditions, including temperature, humidity, andnitrogen can be carefully controlled. The conditions in thisworkstation, as in all others have been defined in the scientistinterface software as the assay is developed. This results in an entryin the workstation operations table (in the data processing andintegration module) for the particular work unit. As the work unit isprocessed, entries are made by the supervisory controller and canoptionally provide an audit trail and error notification of processdeviations for each work unit.

Following incubation 812, the work unit may be transferred to anadditional reagent addition workstation 813. At this point, dyes,substrate, or additional indicators are added to facilitate the detectoroperation. If necessary an additional incubation 814 can be provided. Insome cases this incubation is simply performed as part of the transportoperation to the detector workstation through a measured time delay.

The work unit is then transferred to the detector 815. Detectors canvary depending on the assay and multiple detectors may exist on the samesystem. Typical detectors will measure emitted or fluorescent light, ormeasure a calculated ratio of light from each well of the work unit.After the work unit is processed at the detector, it could betransferred to a waste station, or returned to the storage and retrievalmodule.

FIG. 13 shows a user interaction flowchart that describes a flow fromthe user's point of view. Since both a scientist and operator interfaceare typically provided, the operator of the system is primarilyconcerned with the momentary operations requirements of the automatedunits. A single operator interface is normally used. Typical operationsthat will be included are: (1) compound addition, (2) replenishment, (3)plate generation, (4) defining an assay or procedure, (5) executing ascreen, (6) scheduling work, and when no other operation is necessary,(7) observing system operation.

In some embodiments, it will be desirable to include an incubator. Anexample of such a screening system is shown in FIG. 11. An incubator canhave environmental control of atmosphere (e.g., carbon dioxide andoxygen level for cells), humidity, and temperature. Its capacity andingress and egress points preferably allow for random access byautomation.

It is also desirable to operably link an incubator with a transportmechanism such that any plate can be positioned at a common interfacepoint. A conduit (e.g., linear and orthogonal conduit) transports plateswithin a controlled environment such that as they are requested, theyare presented to a common access point. Empty places within this conduitallow new plates to enter the controlled atmosphere. Environmentalparameters are measured and controlled via proportional integral,derivative controllers with appropriate sensors. Proportional integral,derivative controllers apply mathematical parameters to react to changesin a measured value and generate a control output.

Methods of Identifying Useful Chemicals

The invention includes methods for identifying useful chemicals. Forexample, the method can comprise:

A. retrieving from a storage and retrieval module a plurality ofchemicals in solution in addressable chemical wells, a reagent wellretriever and having programmable selection and retrieval of theaddressable chemical wells and having a storage capacity for at least100,000 of the addressable wells,

B. transporting selected addressable chemical wells with a sampletransporter to the sample distribution module and the sample transporteroptionally having programmable control of transporting of the selectedaddressable chemical wells,

C. aspirating the plurality of chemicals with a sample distributionmodule comprising a liquid handler to aspirate solutions from selectedaddressable chemical wells, the sample distribution module havingprogrammable selection of and aspiration from the selected addressablechemical wells,

D. dispensing the plurality of chemicals with the sample distributionmodule using computer programmable dispensation into addressable samplewells,

E. transporting the addressable sample wells with a sample transporterto a reagent dispenser module and optionally having programmable controlof transporting of the addressable sample wells,

F. dispensing at least one assay solution into the addressable samplewells with a reagent dispenser, and

G. detecting a signal in the addressable sample wells with afluorescence detector.

The storage and retrieval module, the sample distribution module, andthe reaction module are integrated and programmably controlled by a dataprocessing and integration module; and the storage and retrieval module,the sample distribution module, the sample transporter, the reactionmodule and the data processing and integration module are operablylinked to facilitate rapid processing of the addressable sample wells.Typically, the methods can individually screen at least 25,000 selectedand discrete chemicals in addressable sample wells in 24 hours with asingle system.

Preferably, chemical libraries are screened such that at least onechemical library of structurally related chemicals. The chemical libraryis preferably a directed chemical library based on structure activityrelationships or chemistry of a specific chemical moiety. Examples ofchemical libraries and screening (such random library screening) aredescribed herein and known in the art. The system can also be used tocreate pools of chemicals.

Preferably, the transporting step is a parallel transport for parallelprocessing of the addressable wells with the sample transporter. It willbe particularly desirable to include adaptive routing, programmablycontrolled, by the data processing and integration module as part of themethod for transporting plates. The transporting can further compriseseparating the addressable sample wells from contact with the sampletransporter and contacting the addressable sample wells with a X,Ypositioner in a workstation, such as a reagent dispenser module. Thesample transporter can be a conveyor means that can transport platesabout 500 plates per hour across about 3 meters.

The dispensing step can preferably provide for a dilution of about 1,000to 5,000 fold. Dispensing into small volumes facilitates processing.Volumes less than about 5 microliters for each addressable sample wellis particularly useful. The dispensing is into addressable sample wellsthat typically have a well center to well center distance of less than 5millimeters and are disposed on a plate with a standard footprint nolarger than a standard microtiter plate footprint. The dispensingtypically comprises dispensing a predetermined volume of an assaysolution comprising a target into the addressable sample wells. Thetargets can be those described herein, known in the art or developed inthe future, for instance from genomics. Targets include proteins such asmembrane protein and soluble proteins. The dispensing can also comprisedispensing a predetermined volume of an assay solution comprising anagonist, antagonist or other chemical that interacts with the target touse as a positive or negative control, which can help determine thelevel of activity of the test chemical. The signal in the addressablesample wells due to the test chemical can be less or more than thecontrol depending on the control. Activity is often measured from areporter that indirectly or directly indicates the activity of thechemical against the target. In most circumstances, if the reporterproduces less of the signal in the presence of a modulator than in thepresence of the agonist alone, it can indicate that the modulator is anantagonist. In most circumstances, if the reporter produces more of asignal in the presence of a modulator than in the presence of theantagonist, it can indicate that the modulator is an agonist.

Preferably, a plurality of chemicals is aspirated from selectedaddressable chemical wells disposed on a first plate with a firstdensity of wells per cm squared. The first plate is then transportedaway from the liquid handler and addressable sample wells disposed on asecond plate, with a second density of wells per cm squared aretransported to the liquid handler. The second plate is then positionedfor dispensation by the liquid handler. The dispensing of the pluralityof chemicals from the selected addressable chemical wells with theliquid handler then proceeds into a first set of selected addressablesample wells disposed on the second plate. The second plate typicallyhas a higher density of wells than the first plate for screening.

Many different assays can be employed with the invention, such asbiochemical and cell based assays. Fluorescent probes can be substratesfor enzymes, dyes, fluorescent proteins and any other moiety that canproduce a fluorescent signal under the appropriate conditions. Forexample, probes described in PCT application PCT US95/14692 (Tsien), PCTapplication PCT US96/04059 (Tsien), PCT application PCT US96/09652(Tsien), and U.S. Patent application, 08/680,877 (Tsien and Cubitt),U.S. Patent application, 08/706,408, (Tsien) can be used.

In another embodiment, the invention provides a method of developing atherapeutic chemical. The method comprises: (a) testing a chemical formodulating activity of a target, in a device of the invention, (b)testing the chemical against the molecular target for the modulatingactivity determined in step (a), (c) testing the chemical or aderivative thereof against a different molecular target for modulatingactivity, and (d) testing either the chemical or the derivative testedin step (c) in an animal for modulating activity.

Targets

One method of the present invention uses targets for identifyingchemicals that are useful in modulating the activity of a target. Thetarget can be any biological entity, such as a protein, sugar, nucleicacid or lipid. Typically, targets will be proteins such as cell surfaceproteins or enzymes. Targets can be assayed in either biochemical assays(targets free of cells), or cell based assays (targets associated with acell).

For example, cells may be loaded with ion or voltage sensitive dyes toreport receptor or ion channel activity, such as calcium channels orN-methyl-D-aspartate (NMDA) receptors, GABA receptors, kainate/AMPAreceptors, nicotinic acetylcholine receptors, sodium channels, calciumchannels, potassium channels excitatory amino acid (EAA) receptors,nicotinic acetylcholine receptors. Assays for determining activity ofsuch receptors can also use agonists and antagonists to use as negativeor positive controls to assess activity of tested chemicals. Inpreferred embodiments of automated assays for identifying chemicals thathave the capacity to modulate the function of receptors or ion channels(e.g., agonists, antagonists), changes in the level of ions in thecytoplasm or membrane voltage will be monitored using an ion-sensitiveor membrane voltage fluorescent indicator, respectively. Among theion-sensitive indicators and voltage probes that may be employed, arethose disclosed in the Molecular Probes 1997 Catalog, hereinincorporated by reference.

Other methods of the present invention concern determining the activityof receptors. Receptor activation can sometimes initiate subsequentintracellular events that release intracellular stores of calcium ionsfor use as a second messenger. Activation of some G-protein-coupledreceptors stimulates the formation of inositol triphosphate (IP3 aG-protein coupled receptor second messenger) through phospholipase C-mediated hydrolysis of phosphatidylinositol, Berridge and Irvine (1984),Nature 312: 315-21. IP3 in turn stimulates the release of intracellularcalcium ion stores. Thus, a change in cytoplasmic calcium ion levelscaused by release of calcium ions from intracellular stores can be usedto reliably determine G-protein-coupled receptor function. AmongG-protein-coupled receptors are muscarinic acetylcholine receptors(mAChR), adrenergic receptors, serotonin receptors, dopamine receptors,angiotensin receptors, adenosine receptors, bradykinin receptors,metabotropic excitatory amino acid receptors and the like. Cellsexpressing such G-protein-coupled receptors may exhibit increasedcytoplasmic calcium levels as a result of contribution from bothintracellular stores and via activation of ion channels, in which caseit may be desirable although not necessary to conduct such assays incalcium-free buffer, optionally supplemented with a chelating agent suchas EGTA, to distinguish fluorescence response resulting from calciumrelease from internal stores.

Other assays can involve determining the activity of receptors which,when activated, result in a change in the level of intracellular cyclicnucleotides, e.g., cAMP, cGMP. For example, activation of some dopamine,serotonin, metabotropic glutamate receptors and muscarinic acetylcholinereceptors results in a decrease in the cAMP or cGMP levels of thecytoplasm. Furthermore, there are cyclic nucleotide-gated ion channels,e.g., rod photoreceptor cell channels and olfactory neuron channels(see, Altenhofen, W. et al. (1991) Proc. Natl. Acad. Sci U.S.A. 88:9868-9872 and Dhallan et al. (1990) Nature 347: 184-187) that arepermeable to cations upon activation by binding of cAMP or cGMP. Incases where activation of the receptor results in a decrease in cyclicnucleotide levels, it may be preferable to expose the cells to agentsthat increase intracellular cyclic nucleotide levels, e.g., forskolin,prior to adding a receptor-activating compound to the cells in theassay. Cells for this type of assay can be made by co-transfection of ahost cell with DNA encoding a cyclic nucleotide-gated ion channel andDNA encoding a receptor (e.g., certain metabotropic glutamate receptors,muscarinic acetylcholine receptors, dopamine receptors, serotoninreceptors, and the like), which, when activated. causes a change incyclic nucleotide levels in the cytoplasm.

Any cell expressing a protein target in sufficient quantity formeasurement in a cellular assay can be used with the invention. Cellsendogenously expressing can work as well as protein expressed fromheterologous nucleic acids. For example, cells which may be transfectedwith a suitable vector encoding one or more such targets that are knownto those of skill in the art or may be identified by those of skill inthe art. Although essentially any cell which expresses endogenous ionchannel or receptor activity may be used, when using receptors orchannels as targets it is preferred to use cells transformed ortransfected with heterologous DNAs encoding such ion channels and/orreceptors so as to express predominantly a single type of ion channel orreceptor. Many cells that may be genetically engineered to express aheterologous cell surface protein are known. Such cells include, but arenot limited to, baby hamster kidney (BHK) cells (ATCC No. CCL10), mouseL cells (ATCC No. CCLI.3), Jurkats (ATCC No. TIB 152) and 153 DG44 cells[see, Chasin (1986) Cell. Molec. Genet. 12: 555] human embryonic kidney(HEK) cells (ATCC No. CRL1573), Chinese hamster ovary (CHO) cells (ATCCNos. CRL9618, CCL61, CRL9096), PC12 cells (ATCC No. CRL17.21) and COS-7cells (ATCC No. CRL1651). Preferred cells for heterologous cell surfaceprotein expression are those that can be readily and efficientlytransfected. Preferred cells include Jurkat cells and HEK 293 cells,such as those described in U.S. Pat. No. 5,024,939 and by Stillman etal. (1985) Mol. Cell. Biol. 5: 2051-2060.

Exemplary membrane proteins include, but are not limited to, surfacereceptors and ion channels. Surface receptors include, but are notlimited to, muscarinic receptors, e.g., human M2 (GenBank accession#M16404); rat M3 (GenBank accession #M16407); human M4 (GenBankaccession #M16405); human M5 (Bonner, et al., (1988) Neuron 1, pp.403-410); and the like; neuronal nicotinic acetylcholine receptors,e.g., the human α₂, α₃, and β₂, subtypes disclosed in U.S. Ser. No.504,455 (filed Apr. 3, 1990, which is hereby expressly incorporated byreference herein in its entirety); the human α₅ subtype (Chini, et al.(1992) Proc. Natl. Acad. Sci. U.S.A. 89: 1572-1576), the rat X2 subunit(Wada, et al. (1988) Science 240, pp. 330-334); the rat α₃ subunit(Boulter, et al. (1986) Nature 319, pp. 368-374); the rat α₄ subunit(Goldman, et al. (1987) Cell 48, pp. 965-973); the rat α₅ subunit(Boulter, et al. (1990) I. Biol. Chem. 265, pp. 4472-4482); the chickenα₇ subunit (Couturier et al. (1990) Neuron 5: 847-856); the rat β₂subunit (Deneris, et al. (1988) Neuron 1, pp. 45-54) the rat β₃ subunit(Deneris, et al. (1989) J. Biol. Chem. 264, pp. 6268-6272); the rat, β₄subunit (Duvoisin, et al. (1989) Neuron 3, pp. 487-496); combinations ofthe rat α subunits, β subunits and a and p subunits; GABA receptors,e.g., the bovine n, and p, subunits (Schofield, et al. (1987) Nature328, pp. 221-227); the bovine n, and a, subunits (Levitan, et al. (1988)Nature 335, pp. 76-79); the γ-subunit (Pritchett, et al. (1989) Nature338, pp. 582-585); the p, and p, subunits (Ymer, et al. (1989) EMBO J.8, pp. 1665-1670); the 6 subunit (Shivers, B. D. (1989) Neuron 3, pp.327-337); and the like; glutamate receptors, e.g., rat GluR1 receptor(Hollman, et al. (1989) Nature 342, pp. 643-648); rat GluR2 and GluR3receptors (Boulter et al. (1990) Science 249:1033-1037; rat GluR4receptor (Keinanen et al. (1990) Science 249: 556-560); rat GluR5receptor (Bettler et al. (1990) Neuron 5: 583-595) g rat GluR6 receptor(Egebjerg et al. (1991) Nature 351: 745-748); rat GluR7 receptor(Bettler et al. (1992) neuron 8:257-265); rat NMDARI receptor (Moriyoshiet al. (1 991) Nature 354:31-37 and Sugihara et al. (1992) Biochem.Biophys. Res. Comm. 185:826-832); mouse NMDA el receptor (Meguro et al.(1992) Nature 357: 70-74); rat NMDAR2A, NMDAR2B and NMDAR2C receptors(Monyer et al. (1992) Science 256: 1217-1221); rat metabotropic mGluR1receptor (Houamed et al. (1991) Science 252: 1318-1321); ratmetabotropic mGluR2, mGluR3 and mGluR4 receptors (Tanabe et al. (1992)Neuron 8:169-179); rat metabotropic mGluR5 receptor (Abe et al. (1992)I. Biol. Chem. 267: 13361-13368); and the like; adrenergic receptors,e.g., human p1 (Frielle, et al. (1987) Proc. Natl. Acad. Sci. 84, pp.7920-7924); human α₂ (Kobilka, et al. (1987) Science 238, pp. 650-656);hamster β₂ (Dixon, et al. (1986) Nature 321, pp. 75-79); and the like;dopamine receptors, e.g., human D2 (Stormann, et al. (1990) Molec.Pharm. 37, pp. 1-6); mammalian dopamine D2 receptor (U.S. Pat. No.5,128,254); rat (Bunzow, et al. (1988) Nature 336, pp. 783-787); and thelike; NGF receptors, e.g., human NGF receptors (Johnson, et al. (1986).Cell 47, pp. 545-554); and. the like; serotonin receptors, e.g., human5HT1a (Kobilka, et al. (1987) Nature 329, pp. 75-79); serotonin 5HT1Creceptor (U.S. Pat. No. 4,985,352); human 5HT1D (U.S. Pat. No.5,155,218); rat 5HT2 (Julius, et al. (1990) PNAS 87, pp.928-932); rat5HT1c (Julius, et al. (1988) Science 241, pp. 558-564); and the like.

Ion channels include, but are not limited to, calcium channels comprisedof the human calcium channel α₂ β and/or γ-subunits disclosed incommonly owned U.S. application Ser. Nos. 07/745,206 and 07/868,354,filed Aug. 15, 1991 and Apr. 10, 1992, respectively, the contents ofwhich are hereby incorporated by reference; (see also, WO89/09834; humanneuronal α₂ subunit); rabbit skeletal muscle al subunit (Tanabe, et al.(1987) Nature 328, pp. 313-E318); rabbit skeletal muscle α₂ subunit(Ellis, et al. (1988) Science 241, pp. 1661-1664); rabbit skeletalmuscle p subunit (Ruth, et al. (1989) Science 245, pp. 1115-1118);rabbit skeletal muscle γ subunit (Jay, et al. (1990) Science 248, pp.490-492); and the like; potassium ion channels, e.g., rat brain (BK2)(McKinnon, D. (1989) J. Biol Chem. 264, pp. 9230-8236); mouse brain(BK1) (Tempel, et al. (1988) Nature 332, pp. 837-839); and the like;sodium ion channels, e.g., rat brain I and II (Noda, et al. (1986)Nature 320, pp. 188-192); rat brain III (Kayano, et al. (1988) FEBSLett. 228, pp. 187-1.94); human II (ATCC No. 59742, 59743 and Genomics5: 204-208 (1989); chloride ion channels (Thiemann, et al. (1992),Nature 356, pp. 57-60 and Paulmichl, et al. (1992) Nature 356, pp.238-241), and others known or developed in the art.

Intracellular receptors may also be used as targets, such as estrogenreceptors, glucocorticoid receptors, androgen receptors, progesteronereceptors, and mineralocorticoid receptors, in the invention.Transcription factors and kinases can also be used as targets, as wellas plant targets.

Various methods of identifying activity of chemical with respect to atarget can be applied, including: ion channels (PCT publication WO93/13423), intracellular receptors (PCT publication WO 96/41013),U.S.Pat. No. 5,548,063, U.S. Pat. No. 5,171,671, U.S. Pat. No. 5,274,077,U.S. Pat. No. 4,981,784, EP 0 540 065 A1, U.S. Pat. No. 5,071,773, andU.S. Pat. No. 5,298,429. All of the foregoing references are hereinincorporated by reference in their entirety.

Chemicals Discovered by the Operation of Components or Systems andRelated Compositions

The invention includes novel chemicals identified as having activity bythe operation of methods, systems or components described herein. Suchnovel chemicals do not include chemicals already publicly known in theart as of the filing date of this application. Typically, a chemicalwould be identified as having activity from using the invention and thenits structure revealed from a proprietary database of chemicalstructures or determined using analytical techniques.

One embodiment of the invention is a chemical with useful activity,comprising a chemical identified by a system as having modulatingactivity of a molecular target. The system is a device for rapidlyprocessing liquid samples, comprising: (a) a storage and retrievalmodule, comprising storage locations for storing a plurality of reagentsin solution in addressable chemical wells, a reagent well retrieverhaving programmable selection and retrieval of the addressable chemicalwells and having a storage capacity for at least 100,000 addressablewells, (b) a sample distribution module comprising a liquid handler toaspirate or dispense solutions from selected addressable chemical wells,the sample distribution module having programmable selection of, andaspiration from, the selected addressable chemical wells andprogrammable dispensation into selected addressable sample wells and theliquid handler can dispense into arrays of addressable wells withdifferent densities of addressable wells per centimeter squared, (c) asample transporter to transport the selected addressable chemical wellsto the sample distribution module and optionally having programmablecontrol of transport of the selected addressable chemical wells, (d) areaction module comprising either a reagent dispenser to dispensereagents into the selected addressable sample wells for reaction or afluorescent detector to detect chemical reactions in the selectedaddressable sample wells, and (e) a data processing and integrationmodule. The storage and retrieval module, the sample distributionmodule, and the reaction module are integrated and programmablycontrolled by the data processing and integration module; and thestorage and retrieval module, the sample distribution module, the sampletransporter, the reaction module and the data processing and integrationmodule are operably linked to facilitate rapid processing of theaddressable sample wells. The device can process at least 25,000addressable wells in 24 hours. The invention also includes methods ofmodulating targets in cells (e.g., in vivo or in vitro) with compoundsidentified as having modulating activity by the inventions.

The invention also includes compositions comprising a chemicalidentified by systems described herein as having modulating activity ofa target. The composition includes a carrier for the chemical. Mostchemicals in such compositions will be at least 50% pure by weight,preferably at least 80% pure by weight, more preferably at least 95%pure by weight, and most preferably at least 99% pure by weight.Although natural products and combinatorial chemistry products oftenhave purities lower than 80% by weight.

Such chemicals include small organic molecules, nucleic acids, peptidesand other molecules readily synthesized by techniques available in theart and developed in the future. For example, the followingcombinatorial compounds are suitable for screening: peptoids (PCTPublication No. WO 91/19735, Dec. 26, 1991), encoded peptides (PCTPublication No. WO 93/20242, Oct. 14, 1993), random bio-oligomers (PCTPublication WO 92/00091, Jan. 9, 1992), benzodiazepines (U.S. Pat. No.5,288,514), diversomeres such as hydantoins, benzodiazepines anddipeptides (Hobbs DeWitt, S. et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al., J. Amer.Chem. Soc. 114: 6568 (1992)), nonpeptidal peptidomimetics with aBeta-D-Glucose scaffolding (Hirschmann, R. et al., J. Amer. Chem. Soc.114: 9217-9218 (1992)), analogous organic syntheses of small compoundlibraries (Chen, C. et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho, C. Y. et al., Science 261: 1303 (1993)), and/orpeptidyl phosphonates (Campbell, D. A. et al., J. Org. Chem. 59: 658(1994)). See, generally, Gordon, E. M. et al., J. Med Chem. 37: 1385(1994). The contents of all of the aforementioned publications areincorporated herein by reference.

Pharmaceutical Compositions

The present invention also encompasses pharmaceutical compositionsprepared for storage and subsequent administration, which have apharmaceutically effective amount of the products disclosed above in apharmaceutically acceptable carrier or diluent. Acceptable carriers ordiluents for therapeutic use are well known in the pharmaceutical art,and are described, for example, in Remington's Pharmaceutical Sciences,Mack Publishing Co. (A. R. Gennaro edit. 1985). Preservatives,stabilizers, dyes and even flavoring agents may be provided in thepharmaceutical composition. For example, sodium benzoate, sorbic acidand esters of p-hydroxybenzoic acid may be added as preservatives. Inaddition, antioxidants and suspending agents may be used.

The compositions of the present invention may be formulated and used astablets, capsules or elixirs for oral administration; suppositories forrectal administration; sterile solutions, suspensions for injectableadministration; and the like. Injectables can be prepared inconventional forms, either as liquid solutions or suspensions, solidforms suitable for solution or suspension in liquid prior to injection,or as emulsions. Suitable excipients are, for example, water, saline,dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate,cysteine hydrochloride, and the like. In addition, if desired, theinjectable pharmaceutical compositions may contain minor amounts ofnontoxic auxiliary substances, such as wetting agents, pH bufferingagents, and the like. If desired, absorption enhancing preparations(e.g., liposomes), may be utilized.

The pharmaceutically effective amount of the composition required as adose will depend on the route of administration, the type of animalbeing treated, and the physical characteristics of the specific animalunder consideration. The dose can be tailored to achieve a desiredeffect, but will depend on such factors as weight, diet, concurrentmedication and other factors which those skilled in the medical artswill. recognize.

In practicing the methods of the invention, the products or compositionscan be used alone or in combination with one another, or in combinationwith other therapeutic or diagnostic agents. These products can beutilized in vivo, ordinarily in a mammal, preferably in a human, or invitro. In employing them in vivo, the products or compositions can beadministered to the mammal in a variety of ways, including parenterally,intravenously, subcutaneously, intramuscularly, colonically, rectally,nasally or intraperitoneally, employing a variety of dosage forms. Suchmethods may also be applied to testing chemical activity in vivo.

As will be readily apparent to one skilled in the art, the useful invivo dosage to be administered and the particular mode of administrationwill vary depending upon the age, weight and mammalian species treated,the particular compounds employed, and the specific use for which thesecompounds are employed. The determination of effective dosage levels,that is the dosage levels necessary to achieve the desired result, canbe accomplished by one skilled in the art. Typically, human clinicalapplications of products are commenced at lower dosage levels, withdosage level being increased until the desired effect is achieved.

In non-human animal studies, applications of potential products arecommenced at higher dosage levels, with dosage being decreased until thedesired effect is no longer achieved or adverse side effects disappear.The dosage for the products of the present invention can range broadlydepending upon the desired affects and the therapeutic indication.Typically, dosages may be between about 10 kg/kg and 100 mg/kg bodyweight, preferably between about 100 kg/kg and 10 mg/kg body weight.Administration is preferably oral on a daily basis.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition. (Seee.g., Fingl et al., in The Pharmacological Basis of Therapeutics, 1975).It should be noted that the attending physician would know how to andwhen to terminate, interrupt, or adjust administration due to toxicity,or to organ dysfunctions. Conversely, the attending physician would alsoknow to adjust treatment to higher levels if the clinical response werenot adequate (precluding toxicity). The magnitude of an administrateddose in the management of the disorder of interest will vary with theseverity of the condition to be treated and to the route ofadministration. The severity of the condition may, for example, beevaluated, in part, by standard prognostic evaluation methods. Further,the dose and perhaps dose frequency, will also vary according to theage, body weight, and response of the individual patient. A programcomparable to that discussed above may be used in veterinary medicine.

Depending on the specific conditions being treated, such agents may beformulated and administered systemically or locally. Techniques forformulation and administration may be found in Remington'sPharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa.(1990). Suitable routes may include oral, rectal, transdermal, vaginal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections.

For injection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks' solution, Ringer's solution, or physiological saline buffer. Forsuch transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art. Use of pharmaceutically acceptable carriersto formulate the compounds herein disclosed for the practice of theinvention into dosages suitable for systemic administration is withinthe scope of the invention. With proper choice of carrier and suitablemanufacturing practice, the compositions of the present invention, inparticular, those formulated as solutions, may be administeredparenterally, such as by intravenous injection. The compounds can beformulated readily using pharmaceutically acceptable carriers well knownin the art into dosages suitable for oral administration. Such carriersenable the compounds of the invention to be formulated as tablets,pills, capsules, liquids, gels, syrups, slurries, suspensions and thelike, for oral ingestion by a patient to be treated.

Agents intended to be administered intracellularly may be administeredusing techniques well known to those of ordinary skill in the art. Forexample, such agents may be encapsulated into liposomes, thenadministered as described above. All molecules present in an aqueoussolution at the time of liposome formation are incorporated into theaqueous interior. The liposomal contents are both protected from theexternal micro-environment and, because liposomes fuse with cellmembranes, are efficiently delivered into the cell cytoplasm.Additionally, due to their hydrophobicity, small organic molecules maybe directly administered intracellularly.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein. Inaddition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions. The pharmaceuticalcompositions of the present invention may be, manufactured in a mannerthat is itself known, e.g., by means of conventional mixing, dissolving,granulating, dragee-making, levitating, emulsifying, encapsulating,entrapping, or lyophilizing processes.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, rice,starch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Dragee cores areprovided with suitable coatings. For this purpose, concentrated sugarsolutions may be used, which may optionally contain gum arabic, talc,polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dye-stuffs or pigments may be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses.

Computer Program Product, Computational Methods, Databases and StorageDevices Based on Information Generated by the Operation of Components orSystems

In one embodiment, the invention provides for a computer program productfor processing samples and methods. The computer program product caninclude many of the methods described herein such as those methodsrelated to databases, workflow, structure activity relationships,computer interfaces, identification of chemicals, parallel processingand adaptive routing.

For example, the invention includes a computer program product,comprising a computer useable medium having computer program logicrecorded thereon for enabling a computer processor in a system to assistin performing a liquid sample process having a predetermined set ofliquid sample process properties, the system comprising a storage andretrieval module to store and retrieve, in accordance with store andretrieve instructions, a plurality of addressable wells; a sampletransporter to transport, in accordance with transport instructions, aplurality of addressable wells, and a reaction module to react chemicalsor to detect a physical property, in accordance with reaction ordetection instructions, in a plurality of addressable wells. Thecomputer program logic comprises:

A. a workflow model means for enabling the computer processor to definethe process properties of integrated components of the system to enhancedistribution of workflow in the system, and

B. a processing instruction means for enabling the computer processor togenerate processing instructions for routing workflow comprising: 1)store and retrieve instructions, 2) transport instructions, and 3)reaction and detection instructions that, when executed, enable thesystem to rapidly process addressable wells.

In another embodiment the invention includes a database forcomputational analysis of chemical structure and activity againsttargets, comprising activity data of discrete chemicals. The data isstored on a computer accessible storage media. The data is generated bya system that can test for modulating activity of a molecular target.The system is a device for rapidly processing liquid samples, comprisinga storage and retrieval module comprising racks for storing a pluralityof chemicals in solution in addressable chemical wells, a chemical wellretriever and having programmable selection and retrieval of theaddressable chemical wells and having a storage capacity for at least100,000 of the addressable wells, a sample distribution modulecomprising a liquid handler to aspirate or dispense solutions fromselected the addressable chemical wells. The sample distribution modulehas programmable selection of and aspiration from the selectedaddressable chemical wells and programmable dispensation intoaddressable sample wells. The liquid handler of the sample distributionmodule can dispense into arrays of addressable wells with differentdensities of addressable wells per centimeter squared. It also includesa sample transporter to transport the selected addressable chemicalwells to the sample distribution module that optionally has programmablecontrol of transport of the selected addressable chemical wells and areaction module comprising either a reagent dispenser to dispensereagents into the addressable sample wells for reaction or a fluorescentdetector to detect chemical reactions in the addressable sample wells,and a data processing and integration module. The storage and retrievalmodule, the sample distribution module, and the reaction module areintegrated and programmably controlled by the data processing andintegration module; and the storage and retrieval module, the sampledistribution module, the sample transporter, the reaction module and thedata processing and integration module are operably linked to facilitaterapid processing of the addressable sample wells. The device can processat least 25,000 addressable wells in 24 hours. Other database relatedembodiments are contemplated with other systems described herein, aswell as methods using the databases and storage devices storing thedatabase.

EXAMPLES Example 1

Screening Sample Distribution Module

The screening sample distribution module permits the preparation ofplates with chemicals, such as test chemicals from addressable chemicalwells and biological reagents (e.g., cells or isolated moleculartargets). The primary function of the screening sample distributionmodule is to aspirate solutions from one plate and transfer them intoanother plate. This is usually accomplished with an array of liquidhandlers (e.g., liquid handling head), preferably an array of at leastabout 50, more preferably at least about 100 and most preferably atleast about 200. The array is most preferably M liquid handlers by Nliquid handlers, wherein M is the number of addressable wells in acolumn on a plate or an integer multiple thereof and N is the number ofaddressable wells in a row on such plate or an integer multiple thereof(wherein M and N preferably have the same integer multiple), asdescribed herein. The sample distribution module can be integrated in ascreening system, for example, as shown in FIG. 1 with a data processingand integration module. The screening sample distribution module can beoperably linked to other workstations with a sample transporter, whichis also shown in FIG. 2. The sample distribution module for bothaspiration and dispensing in one embodiment comprises, dispensers,stackers, a liquid, a reader and a conveyor.

Dispensers

In one embodiment the screening sample distribution module was designedwith a liquid handler, having. 96-dispensers that can use positivedisplacement disposable tips in 200 μL, 50 μL and 20 μL volumes. Adisposable 200 μL tip head can deliver a range of volumes of 1 μL to 200μL, with precision and accuracy of 10% at 1 μL to 3% at 200 μL. Anoptional disposable 20 μL tip can deliver a range of volumes of 0.1 μLto 20 μL, with precision and accuracy of 10% at 0.1 μL to 3% at 20 μL.The dispenser can move in Z-axis (vertical axis, i.e. perpendicular toplane of a floor), which is controlled with a Z-positioner and has atravel distance from below a conveyor, to access a wash station orreagent trough, to above the conveyor. Preferably, a lidded deep wellplate can pass underneath (about 3 to 5.5 cm in distance). The dispenseaxis (the axis with respect to volume displacement) will be at least10,000 steps (servo motor) to displace the full pipetting volume.Dispensing speeds are controlled by positive movement of a shaft and arecontrollable from 1 mm/second to 50 mm/second. The resolution of thez-axis will be at least 25,000 steps over 75 mm travel from below theconveyor to a fully retracted position. Positional feedback may berequired for both z and d-axis (dispense axis), such as encoders, liquidlevel and limit switches. Both axes will be capable of simultaneous andconcurrent operation independent of each other. The dispenser assemblymust be continuously adjustable (no detentes or stops) in “X” and “Y”with a +/−10.0 mm positioning capability with respect to a plateconveyor. Dispensers can accommodate a flowing wash station and arefilling reagent trough. The dispensers can accommodate 384-wellplates, well as 96-well plates. Dispensers can be a piezo device or asolenoid described herein or known in the art or developed in thefuture.

The liquid level for both aspirating and dispensing can be monitored byplacing a sensor on or near the tip of the liquid handler, such as anelectrical sensor. For example, the capacitive sensor described in U.S.Pat. No. 5,365,783 (Zweifel) can be used, as well as other suitablesensors known in the art. Such methods can also be applied to otherliquid handling devices described herein.

Stacker Magazines

In one embodiment, the screening sample distribution module was designedwith a stacker magazine having a capacity of about 50 standardmicroplates. Bi-directional stacking with lidded or unlidded plates isdesirable. The stacker magazine can accommodate either standard heightplates or deep well plates in a given stack and plate types willtypically not be mixed in a stack.

Lidder

In one embodiment, the screening sample distribution module was designedwith a bidirectional plate delidder and relidder. The lidder removes andreplaces plate lids at a rate of about 5 to 11 plates per minute in onedirection. The lidder can store approximately 60 lids. Preferably, amodified lid is used for separation.

Bar Code Reader

In one embodiment, the screening sample distribution module was designedwith a bar code reader to scan incoming plates and verify plate locationin the screening sample distribution module and to permit location of anaddressable well. Typically, bar codes are detected to locate platesbefore any manipulation is performed. Bar code labels were positioned onthe narrow end of the plates, column 12 side, and will be 3 to 1 ratio,0.25″×1.0″, 10 mil bar code 128, from Intermec, Everett, Wash. Misreador unreadable labels will produce an error code available to thesupervisory control system. Labels and adhesive must resist the actionof solvents and environmental conditions.

Conveyor

In one embodiment, the sample distribution module was designed with abi-directional conveyor transport plates within the sample distributionmodule. It can be optionally used to transport plates for movementbetween module components (e.g., dispenser head assembly and stackermagazines). The conveyor operates at a speed allowing a transit timebetween modules of 2-4 seconds or less. Gates are available forarresting plates at predetermined module positions. Detaining of a plateon the conveyor by a gate will not cause the plate to vibrate or movesuch that the module associated with that position fails to perform itsfunction. Conveyors for a sample distribution module are preferablybelt-driven and allow a rotating belt to contact a plate bottom.

Interfaces/Communication

In one embodiment, the screening sample distribution module was designedwith a (programmable logic controller with ladder logic) having aminimum of two RS-232 ports open for external communication andprogramming (or GPIB interfaces). The programmable logic controller withladder logic will have an ethernet communication card enabling theprogrammable logic controller with ladder logic to communicate withanother computer via transmission control protocol/internet protocol.The programmable logic controller with ladder logic will have fourdiscrete inputs and three discrete outputs open for handshaking to aconveyor system. The conveyor control logic is written to conform to thehandshaking logic outlined in FIG. 6. The sample duplication robotE-Stop switch must be a dual pole design to allow an external E-Stoploop to be wired through the switch.

Error Conditions

Failure of the sample distribution module to perform any programmedfunction within an allotted time will also constitute an error.Performance of a function outside of measurable parameters will alsoconstitute an error. Errors will be corrected automatically when withinthe ability of the instrument to do so. Unrecoverable errors can notifythe user via both the touch screen and the external link, and will setthe handshaking logic to refuse further plate input, until the error iscorrected. For given families of error conditions, a response can bespecified, e.g., for recoverable errors, bar code errors. For errorsthat are automatically recoverable, response parameters will exist toeither pause the instrument and report the error, or to automaticallyrecover from the error, report/log the error and resume operation.Recoverable errors will have a time-out function to halt recovery, iftime exceeds a configurable value.

Functional Capability

The control software can control the parameters as specified in Table 3,via an interface. Level two will facilitate the same function via anexternal computer.

TABLE 3 Bar Code Conveyor Delidder Aspirate Aspirate Range DispenseDispense Range Stacker Read ON/OFF Up/Down Z Height (mm) −30 to 60   ZHeight (mm) −30 to 60   Up/Down Compare Forward/ Volume (μL)  0 to 200Volume (μL)  0 to 200 Reverse Exception Speed (%)  0 to 100 Speed (%)  0to 100 Overfill (μL)  0 to 200 Air Gap (μL)  0 to 200 Pre-Dispense (μL) 0 to 200 Plate/Bath   0 or >0

Replication/Expansion (Master to Multiple Daughters) A master plate canbe distributed into one or more daughter plates. The master plate isaspirated from one or more of the pipetting stations in the sampledistribution module. Daughter plates are positioned under thesepipettors. If the replicate volume is large, e.g. volume×replicatenumber is greater than the tip volume, then multiple aspirations fromthe master are required. Additionally, it is often faster to bring infour masters and then four daughters (one for each master) and repeatthis until each master is replicated completely. The master may also be384 and the replicates 96-well plates. If this is the case, the onlydifference is that the master must travel under each pipettor to accesseach quadrant. Later the master could be 864-well and have ninedaughters produced.

Pooling/Compression (Multiple Masters to a Single Daughter) Multiplemaster plates are positioned under separate pipettors. A single daughteris dispensed to by each pipettor. This may be in the same wells orseparate wells of a 384-well plate or greater density. Pooling couldconsist of more than four masters being combined to a single daughter,this would require the daughter to be sequestered while new masters wereaspirated from. Later this could be at least 36 masters to one3,456-well plate.

Example 2

Plate Handling and a Sample Transporter

The screening system typically operates on a work unit (unit) with astandardized processing format. The work unit sometimes embodies aparticular processing format, such as a plate. Such work units oftenvary in external dimensions, materials of construction, physical designand density (or number of wells). The screening system is preferablyconstructed using workstations to handle a standard processing formatthat allows for a flexible work unit. This approach reduces the need forcomplete uniformity of the work unit, as is required in many laboratoryautomation and discovery systems. Consequently, a screening system canfunction with a mixture of many work units without adjustment orcalibration. This permits the system to universally handle varying workunits of a standardized processing format. The sample transporterpreferably comprises components that permit universal plate handling.The sample transporter can be used to operably link workstationstogether as shown in FIG. 11.

Typically, a storage and retrieval module and operably linked sampletransporter include a storage and retrieval end actuator, universalstacker/destacker, transport system elementsand the registration devicethat are designed to handle work units with a standardized processingformat. Preferably, to enhance workflow and throughput, these all thedevices that handle work units that are designed to handle the samestandard plate footprint.

FIG. 14 shows part of a storage and retrieval end actuator with part ofa storage and retrieval module. The storage and retrieval end actuatorcan be used as a universal end actuator and adapted to move a work unitwith a standardized processing format. For example, an storage andretrieval end actuator can be a component of the storage and retrievalmodule to retrieve wells. A design preference of the storage andretrieval module is to maximize the number of units that can becontained in a given vertical and horizontal space. It is undesirable toallow access space on either side of the work unit. The storage andretrieval end actuator provides the advantage, compared to manyactuators, of not requiring any horizontal space to move a plate.

To retrieve a work unit from a storage and retrieval module, the storageand retrieval end actuator can be positioned in front of a particularhotel within the storage and retrieval module using an X and Zpositioner. In FIG. 14, the platen 701 is positioned within a notchedhotel shelf 702 in the hotel 708 enclosure, but slightly below the workunit 704. A drive means 703, extends or retracts push/pull hooks 706 tothe end of the platen under the work unit. At this point, the X and Zpositioner raises the platen a small amount, engaging the hooks beneaththe work unit 704. Work units should have an opening(s) that will engagethe hooks. A push/pull means operates to pull the work unit onto theplaten. Its travel allows the unit to be centered on the platen for thesubsequent upstack operation in the end actuator's stacker 707. Guidescan be fitted, which are not shown, to center the unit on the platen. Anadvantage of the storage and retrieval end actuator is that it canoperate on any work unit that has an opening, such as a concave orhollow bottom found on commercially available plates. The storage andretrieval end actuator does not require any other physical dimension orspecial tooling of the work unit, although it may be preferred.

The placement of a work unit in the storage and retrieval module (or ona sample transporter) is similar, except the push/pull hooks arepositioned to allow the push/pull hooks to push the work unit by itsfront edge into the notched hotel shelf. To store a work unit, theplaten is positioned slightly above the floor of the notched hotelshelf. The storage and retrieval end actuator can be optionallyconfigured with a detector that identifies a work unit by its bar codeor other identifying marking.

A universal stacker/destacker that can handle work units, e.g., plates,by stacking or destacking a plurality of units, both with and withoutlids. It can often act as a buffer between an storage and retrievalmodule and the sample transporter. The universal stacker/destackershould minimize the height of a completed stack. Ideally, stack heightshould not be any more that the sum of the height of the stacked units.The universal stacker/destacker should also be able to accept plates ofdifferent heights. Plates can vary in height depending on the wellcapacity (typically between 1.2 cm and 5.5 cm). Therefore, a separatordevice is largely unacceptable. Stacking and destacking can beaccomplished by a plate lifter that can bottom load and bottom unload aplate in a stack, while maintaining the position of the stack.Alternatively, the universal stacker/destacker may move down or up andthe platen maintain its Z-position. Plates are kept in place by eitherproviding a plate holding means disposed on the universalstacker/destacker frame or a plate holding means disposed on a platen(e.g., as depression on the platen into which a single plate falls whileleaving behind the remainder of the stack when the platen is withdrawn).Preferably, the universal stacker/destacker should also be able torelease an entire stack, if transport of the entire stack is desired.

FIG. 15A (plan view) and FIG. 15B show a Universial stacker/destackerwith a work unit 710 (shown with a lid) on a platen 715. To stack thework unit the platen is vertically lifted into the frame of theuniversal stacker/destacker. The amount of vertical lift distance can bea calculated distance based on the type of work unit. This value canthen be used by the data processing and integration module to controlthe vertical lift of the platen. This information can be contained inthe data processing and integration module Data Store and can be used todrive a work unit's unit Z-positioning device. The work unit is guidedby a three sided stack enclosure 711. As the work unit is lifted, it maycome in contact with pressure pins 714 either indirectly (with anotherwork unit above it that is engaged by pressure pins 714) or directly, ifno work unit is above it. Once the work unit is lifted sufficiently,pressure pins 714 release the work unit immediately above the newlyentered work unit and engage the new work unit. The new work unit, andall higher stacked units 712 are then raised a calculated distance tosecurely engage the lower skirt of a plate 713 with pressure pins 714.The pressure pins can engage the work units with a preset pressure baseda retractable spring or spring like mechanism or a variable pressurebased on a retractable piston that is computer-controlled to permitrelease or engagement of work units.

Down stacking is accomplished in a reverse manner. Pressure pins areactuated and released by compressed air. Lid information is included inthe calculations, so that there is never interference with lidded units.The entire stack may be transferred to the input/output station byreleasing the pressure pins 714 and allowing the platen 715 to transferthe entire stack.

FIG. 16A (side view) and FIG. 16B (plan view) shows the transport systemelements 721 in a section of track that can be used in a sampletransporter. Transport system elements contact and operate on the bottomof work units 720. Work units are transported on a series of poweredtransport system elements (e.g., rollers) 721. Typically, at least fiverollers are in contact with the work unit 720 during normal transfer.Spaces 722 can exist between the rollers to permit sensing devices orphysical actuators to permit sensing or actuation while minimizingdisruption of the transfer of work units. Devices can includemechanically actuated stops 723 to stop or direct plates. Brakes 724 canalso be provided to stop the rotation of some rollers 721 by engagingthe rollers from below. Entire rollers 725 can be eliminated to provideaccess for a larger sensory apparatus, without reducing the transportefficiency, since the unit can be conveyed on its edges, a flat bed ofrollers is not required. Rollers can be eliminated in the center 726 sothat the entire bottom surface of the unit, other than its edges, can beacted upon. This can accommodate identification and detector devices.The elimination of rollers (e.g., rollers that do not span the track) orspaces between rollers in a location can be used to dispose aZ-positioner that engages a work unit from its side or bottom. TheZ-positioner lifts the work unit off the roller for further positioning(e.g., positioning in a workstation) or lowers the unit onto the rollers(e.g., from a work station).

Rollers are preferably made of a material that minimizes friction with awork unit bottom to minimize the force required to transport a work unitwhile providing sufficient friction to prevent interfering slippage andto provide accurate stopping at desired locations. Typically, rollerswill be made of a polymer relatively inert to solvents, preferably aTelfon™ or Delrin™ is used. Roller friction can be adjusted for certainplates by scoring the surface of the roller with tiny striations. As thenumber of striations, or the thickness of striations increases, thefriction between a roller and work unit increases. Striations should beso large as to not significantly disturb the contents of an addressablewell, unless agitation of the wells is desired. Rollers can be used asdescribed in the electronics manufacture arts and as developed in thefuture for the electronics and other industries. The application of suchrollers from the electronics industry to screening and laboratoryautomation applications, however, is unique.

Example 3

Hit Profiler

A screening system can include a hit profiling robot (HPR) to preparetest chemicals that show activity in a screen, i.e. hits. The primaryfunction of the hit profiling robot is to reformat “hits” from sourceplates (e.g., a master or daughter plate) into secondary (ordestination) plates for confirmation of activity and profiling the hit,such as an EC50. Typically, the hit profiling robot is capable ofhandling 96 and 384-well plates, and preferably 3,456-well plates, andit has liquid handling heads that can aspirate from standard height anddeep well plates. A delidder/relidder can be included to remove andreplace lids on plates with lids prior to, and after, aspiration. Asshown in FIG. 2 the hit profiling robot can be integrated to otherworkstations using a data processing and integration module and operablylinked to other workstations with the sample transporter.

The hit profiling robot can be controlled by either a computer thatallows for selection of pre-defined methods by end users, or end usersmay create an entirely new method. Typically, a plurality of addressablewells containing the desired chemical are selected, at least one liquidhandling head is directed to aspirate a solution from an addressablewell and dispense the solution into at least one other addressable well,such as a well of a secondary plate. For most applications the number ofsource plates used to produce a secondary plate will vary and the hitprofiling robot can be designed to hold multiple source plates in astacker. Typically, source and destination plates will have differentheights and densities. To aspirate and dispense into plates of varyingheights the hit profiling robot has liquid handling heads mounted on aZ-positioner or, alternatively, the plates can be securely retained on aZ-positioner and the liquid handling heads held in place. The computerthat controls the hit profiling robot can also report of success,failure and errors related to the hit profiling robots function.

FIG. 17 shows an HPR with articulated 4-particular dispensers 1210 thatcan use positive displacement dispensers. The dispense axis is comprisedof at least 3,000 steps over the full pipetting volume using a servomotor. The resolution of the X-axis is at least 100 microns and isachieved by an X,Y positioner. Positional feedback is used for all axesand achieved with computer-controlled X,Y positioner and Z positioner.This type of control allows for single wells to be accessed, by movingunused heads out of position. Pipetting speeds are electronicallycontrolled. The liquid handling heads can be suitable liquid handlingdevices described herein, including solenoid and piezo based devices, aswell as an other low volume liquid handling devices known in the art ordeveloped in the future. Pipetting volumes typically range from 1 to2,000 nanoliters, and picoliters volumes can be achieved with piezoheads (e.g., 10 to 500 picoliters). The liquid handling head canoptionally sense the liquid level for both aspirating and dispensingusing either capacitance or resistivity sensors. Such sensors canprovide minimum tip exposure to liquids. Tips are optionallyreplaceable. Single undiluted samples from one or more wells of thesource plates are transferred to the destination plate. Dilution seriescould be generated from the source plates into the destination plate.Stackers 1230 can be included and accessed with a conveyor 1220.

A flowing wash station and refilling reagent trough can also beincluded. This will reduce carryover, which is usually no greater than100 ppm. The volume, duration and flow for probe washing arecomputer-controlled.

The hit profiling robot can include an identification code system, suchas a bar code reader to scan incoming plates. The bar code reader 1240is located to read plates before any other manipulation is performed andbar code identification is confirmed upon plate arrival.

Plates are transported to the hit profiling robot using a bi-directionalconveyor, which can operably link the hit profiling robot to othermodules (see FIG. 2). The conveyor can operate at a speed that providesfor a transit time between modules of 2.5 seconds or less. Gates canarrest plates at predetermined module positions. Gates arrest plates onthe conveyor with minimal vibration and movement to prevent the platefrom being transferred improperly to a module. A movement function willallow a destination plate to be “parked.” The hit profiling robot willintegrate into a conveyor transport system using a means fortransferring a plate to the hit profiling robot. Alternatively, the hitprofiling robot can work in a stand alone fashion.

In operation the hit profiling robot can perform simple reformatting ofplates (i.e. transferring solutions from one plate density to another),reformatting with dilution, and reformatting with pooling. For example,the hit profiling robot can transfer solutions from multiple sourcepates to a single destination plate. Alternatively, the hit profilingrobot can perform pooling (or compression) of multiple master plates toa single daughter plate. Multiple master plates are positioned underseparate liquid handling heads and a single daughter plate is made byeach liquid handling head. This may be in the same wells or separatewells of a 384-well plate. For instance, pooling could consist of morethan four master plates being combined into a single daughter plate.This requires the daughter plate to be sequestered while the masterplates are being aspirated.

Example 4

High Capacity Stacking System (HCSS)

The screening system preferably includes a high capacity stacking system(HCSS) that can act as a plate buffer (buffer). The HCSS comprises atwo-dimensional array of plate stacks. Plates are accessed from thebottom. The lowermost plate can be randomly accessible by positioningmultiple stacks over a plate transport conveyor and upstacking ordownstacking from a first stack to a second stack (e.g., shufflingplates between stacks to retrieve a desired plate). The high capacitystacking system provides a 500 plate capacity when implemented with a 2(plate transport conveyors)×5 (stacks) array of 50 plate stacks. Thehigh capacity stacking system in a preferred embodiment is used as aninput source and output buffer, for a liquid processing system (see FIG.11). Alternatively, the high capacity stacking system can be used as aplate conversion device for a screening sample distribution module or ahit profiling robot.

The high capacity stacking system can be operated to perform manydifferent plate sorting functions for screening to improve adaptiverouting or flexibility in screening protocols. For instance, atechnician loads 500 empty plates into the ten stacks of a high capacitystacking system attached at a lift and transport station along thesample transporter (see FIG. 11). The storage and retrieval moduleretrieves master plates from the storage and retrieval module and thehigh capacity stacking system provides empty plates for replication atthe screening sample distribution module at another lift and transportstation on the sample transporter. The resulting daughters are directedback into the high capacity stacking system to be removed by thetechnician. The removable stacks allow a family of workstations to shareinput and output formats, i.e. the stacks are interchangeable betweenworkstations to allow workstations to be linked.

Example 5

Random Access High Density Plate Presentation Module

The random access high density plate presentation module presents highdensity plates to the sample transporter (see FIG. 11). The randomaccess high density plate presentation module can be mechanically linkedto a sample transporter using a platen that positions the plate on thesample transporter by moving the platen partially over the sampletransporter and sliding the plate onto the sample transporter with amember on the platen that slides the plate off the platen and onto thesample transporter. To avoid the plate from being slid onto the sampletransporter, the random access high density plate presentation modulecan include a lifting platen that can be disposed on the sampletransporter to engage the platen or the plate and lift the plate fromthe platen. At which point the platen is withdrawn and the liftingplaten is positioned to allow the plate to rest on the sampletransporter. The random access high density plate presentation modulecan also be mechanically linked to a sample transporter with a conveyormeans. The random access high density plate presentation modulecomprises a plurality of stacks, preferably removable stacks, that holdplates. Each stack is connected to either a platen or a conveyor systemthat has a lifting platen that either stacks or destacks plates byengaging the plate bottom. Alternatively, stacking systems in the priorart can be used, such as those available from Carl Creative Systems(Carson City, Calif., USA) and Packard Instrument Company (Meriden,Conn., USA), or stacking systems developed in the future.

Example 6

Screening Reagent Dispensing Robot

The screening reagent dispensing robot can dispense reagents necessaryfor performing a screen. A screening reagent dispensing robot canrapidly, accurately and reproducibly dispense solutions in anaddressable well in predetermined volumes. In most embodiments thescreening reagent dispensing robot is an array or plurality ofdispensers that are in fluid communication with a reagent reservoir.Aspiration by the dispensers is usually not required. A screeningreagent dispensing robot can be adapted to dispense a particular type ofreagent or, depending on the reagents, different reagents. Usually,washing steps will be required when reagents are switched to minimizecross contamination. Reagents for a screen can include reagent buffers,dyes, agonists, antagonists, and cells. Multiple screening reagentdispensing robots can be integrated and operably linked as part of ascreening system, such the one shown in FIG. 11.

In one embodiment the screening reagent dispensing robot comprisescomputer-controlled electronic valve drivers for precise control ofvoltage pulse width, voltage amplitude and back EMF dissipation. Inapplications where fast delivery speed (e.g., at least about 50 to 1,000microseconds) is important, fast operating valves should be chosen tocontrol fluid flow. This is especially advantageous at the lower end ofdispensed volume range, which often necessitates high resolution,accurate and reproducible control of the valves. Preferablyelectronically controlled (e.g., solenoid) valves are used instead ofpneumatic valves. In such cases, the software in the programmable logiccontroller or computer has a microsecond resolution and accuracy. In oneembodiment the inventors utilized a hardware timed driver that wascapable of simultaneously delivering up to 200 volts to up to 96 valveswith microsecond accuracy and programmable time control.

Measured and calculated dispense times are shown in Table 3 for a lineararray of 48 dispensers.

TABLE 3 Per Dispenser Per 48-Array Per Plate Per Dispenser DispensePositioner Pause Total Volume Time Move time Time Total Time (nanoliter)(msec) (msec) (msec) (msec) (sec) 20 nanoliters  1 500 100 601 43  2microliters 100 500 100 700 50  5 microliters 250 500 100 850 61

The dispenser was a solenoid dispenser with a dynamic range of about 5nanoliters to 10 microliters. The fluid pressure was about 7 psi. At oneminute per plate, a linear array dispenser can fill approximately thirty3,456 plates per hour. This assumes a worst-case time of about 1 minuteto change plates. Thirty such plates is equivalent to over 90,000samples per hour. Assuming 10% of the plates are used for controls,720,000 samples can be processed in an 8 hour work period (1,080,000samples can be processed in a 12 hour work period).

Preferably, solenoid valves are used for a screening reagent dispensingrobot. These valves are capable of delivering as little as 5 nanoliterswith the proper tip configuration. With the caliber tips describedherein, the minimum dispensed volume is in the range of about 25nanoliters. The maximum dispensed volume is mainly limited by the sizeof the reagent reservoir. Comparable valves can be used for preferredembodiments, such as those known in the art or developed in the future.Alternatively, piezo based dispensers can be used to dispense picolitervolumes (e.g., about 5 to 500 picoliters and preferably about 100picoliters). Such piezo devices are available from sources describedherein.

The screening reagent dispensing robot can also include dispenser tipsspecifically designed to accommodate manifolds that permit close spacingof tips. Typically, tips are spaced less than about 5 mm apart andpreferably about less than 1 mm apart. Since fluid connections (e.g.,channels) to the tips are preferably larger than the spacing betweentips, so as to increase fluid flow, minimize hydraulic resistance, andreduce clogging. Fluid connections are disposed in a pattern that allowsa properly angled tip to have a distal tip end disposed with the desiredspacing. Typically, the fluid connections will be staggered along anaxis and the tips will engage the fluid connections at an angle thatpermits the distal tip end to be disposed along the axis at the desiredspacing. Preferably, the tips must allow dispensation within a 1 mminternal diameter well and to produce a stream significantly less than 1mm in diameter in order to not form bubbles as the reagent is deliveredinto the well. In addition, the dispenser tip typically permitsrepeated, individual dispenses of 100 nanoliters.

Tips can be engaged to fluid connections and angled using a variety ofconfigurations. For example, the outlet end of the valve is a stainlesssteel tube 0.2″ (5.08 mm) in length with an O.D. of 0.020″ (0.51 mm) andI.D. of 0.010″ (0.25 mm). This will mate with a 1.25″ (31.75 mm) long,type 304 stainless steel tube (gauge 33, O.D. of 0.008″ (0.20 mm) andI.D. of 004″ (0.10 mm)). The gauge 33 tube fits inside the valve inlettube and the two tubes are bonded together using Teflon heat shrink/melttubing (Small Parts, Inc. cat #: E-SMDT-036). A 25° bend exists 0.5″from the dispensing end of the tip. This 0.5″ inch segment is placed ina tip block which aligns each tip into a linear array. The remaining0.75″ segment is bent 10°, either up or down, to align it with a valvein one of two valve blocks.

A stainless steel block can be used to hold and position the dispensertips into a linear array with a 1.5 mm pitch. Each tip fits into a slotwithin the block. The block has a U-shaped groove that intersects eachslot. Once all the tips are in place and aligned, a holder bar isscrewed into the groove that presses against each tip and holds them inplace.

A stainless steel block can also hold and position valves into a lineararray with a desired pitch. For example, a 6.0 mm pitch would becompatible for a 48 head array using 12 lee valves. Each valve slidesinto a cylindrical groove within the block and is held in place by thetip block's hold on the tip. Four of these valve blocks, placed side byside at an angle and offset 1.5 mm in relation to each other, can yielda linear arrangement of 48 valves tips. If fast flush solenoid valvesare used, it is desirable have fluid connection with the tip via Teflontubing.

The screening reagent dispensing robot has a fluid path to each tip andvalve with minimal volume and dead spaces. Preferably, the fluid path toeach tip is identical (but not necessarily shared) as to otherdispensers. The tip has the most resistance to fluid flow so its designis preferably identical as possible for each dispenser site in order tominimize variation in volume dispensed between valve/dispenser tips. Thevalving system allows residual reagent to be washed prior to delivery ofa new reagent. All fluid lines are preferably Teflon™ (e.g., FEP) orsilicone, or a chemically inert material. The only exception to this isthe tip, which is typically high quality polished stainless steel. Fluidmanifolds are usually constructed in Teflon and mate with the valveinlets via friction fittings. A fluid manifold is usually shared among aplurality of valves, e.g., it connects to 12 valves. The manifold hasports on both ends that allow it to be a flow through or dead endsystem. All the valves can be connected to a single reagent reservoir byconnecting manifolds together in parallel or in series.

The fluid reservoir can vary depending on the application and therequired volumes and solvents. A variety of volume ranges are possibledepending on the type of reservoir that is used. A pressure chamber,like the one from EFD (Model 615DTH), that will allow a variety ofreagent bottles to be used is advantageous, since the whole bottle isplaced inside a pressurized chamber. An alternative is to pressurizejust the bottle and place the bottle within a shielded safety chamber. Athird alternative is to use a fluid reservoir designed to hold lowpressure.

If live cell cultures are being dispensed, the recommended reservoir isa stirred container. An example is the stirred filtration container(Amicon, MA, USA) which has the further advantage of having a built-instirring mechanism to keep cells suspended. This container is availablein 3, 10, 50, 200 and 400 ml sizes. If the dispense routine requires anysignificant amount of time (more than a few minutes), it will benecessary to provide re-circulation of the cell culture through thefluid system in order to prevent adhesion or pooling of the cells. Thiscan be done via peristaltic pumps, though rotary piston pumps may bepreferable since they potentially cause less damage to cells.

Fluid is delivered to the valves by pressurizing the reagent reservoirwith a fluid, such as a gas. The Model 8310 (0-10 psi) pressureregulator from Porter Instrument Co. can be used and the like. HoneywellMicroswitch pressure sensors will monitor pressure and a pressure reliefvalve, rated for 10 psi, and the like can be included. For superior,pressure control and dispensing valves can be arranged in series andelectronically controlled to coordinate dispensing and to providedesired pressures. This can allow pressures to be changed in the fluidsystem if so desired.

The dispenser arrays are typically positioned over the desired wellswith an X,Y positioner. A suitable X,Y positioner preferably, permitsthe array to be positioned over wells having a density greater or lessthan the distance between tips. This allows the screening reagentdispensing robot to be used for plates of different well densities.

FIG. 18 shows different well densities in relation to different tippositions of a dispenser array. A well 1810 is addressed by a possibletip 1820 (filled circles). Also shown are tip positions 1830 (opencircles) that may or may not address a well depending on the welldensity. Each bank shown is an array. The increased spatial density ofthe dispenser and the increased two dimensional density of the targetplate require substantial positioning accuracy. The positioning accuracytolerance is typically about 200 microns or less, and preferably about50 microns or less in order to ensure that the proper position in thewells for dispensation can be achieved. Alternatively, such positioningparadigms can be used for detectors.

A reagent dispensing robot can include integrated computer control formanaging and directing the entire dispense operation. The linear arraydispenser and its integrated positioning requirements can usesophisticated computer control for effective operation. The computer notonly monitors the status of key sensors (e.g., reagent bottle pressure,liquid level, plate position, and positioning limit switches) but alsoprovides the interface for generating specific liquid dispensationpatterns and volumes to the high density plate. Timing of dispensationcan be accomplished by a variety of means known in the art and developedin the future, so long as such timing means are suitable for the timeframe and control desired. For example, the National InstrumentsAT-MIO-16XE-50 board can be used as timing means to send timing signalsto two of their AT-DIO-32F boards. The 64 ports on these 32F boards arekept normally high and send out timed low signals. An inverter board isused to make the timed portion high and these high signals are used toclose high voltage relays (Opto ODC5A) which run the valves. An OV'Rdriver (Lee Company cat # DRVA0000010A) is used to protect the valvesfrom overheating during prolonged open periods.

The software controlling the valves (or dispensers) can be written tointegrate into a screening system or for a standalone use. Software forlaboratory instrumentation is known in the art and can be used. Forexample, software can be written in LabVIEW (National Instruments, TX,USA). The user selects a valve opening time and the valves to be opened.This program can be embedded within a larger program that controls otherfeatures (such as the X,Y positioner) to obtain an automatic dispenser.

All materials must be substantially compatible with the requiredreagents over the time frames that the materials are used. The reagentsshould be relatively inert to components and the materials used in theconstruction of this dispenser must relatively inert to the reagents.This is especially critical for whole viable cells which may be used inscreening assays. Materials are preferably non cytotoxic, non hemolytic,non-aggregating surfaces, and non sticky to biological materials.

The motive force for the liquid can be supplied by pressurized bottles(in a preferred embodiment) or via positive displacement means includingsyringes, pistons, peristaltic and rotary pumping mechanisms. In-linefiltration or inlet filtration can be introduced to the system forreducing contaminating particles.

A preferred embodiment of a screening reagent dispensing robot is a setof at least two linear arrays (banks) of dispensers on a X,Y positionerin fluid communication with a plurality of electronically controlledvalves (e.g., solenoid or piezo) that are in fluid communication with atleast one reagent reservoirs. Dispenser tips are spaced to accommodatethe density of the addressable wells on a plate. For example, for a highdensity plate, tips are about 1.5 mm apart. The X,Y positioner permitseach tip in a bank to be position over the desired well, preferablyindependently of the other banks. The valves are individually andelectronically controlled and can be opened simultaneously or in anypattern with microsecond resolution. Typically, the dispensers canrapidly dispense about 50 to 10,000 nanoliters (or lower ranges about 25picoliters to 1,000 nanoliters) of a single reagent into predeterminedcombination of wells of a high density plate, preferably withoutphysical contact. Preferably, the screening reagent dispensing robot iscompatible with different dispensing solutions, including aqueous,alcohol and DMSO based reagents.

FIG. 19A (cut-away view of assembled unit) and B (exposed view of onebank) shows components of one embodiment of a screening reagentdispensing robot that includes valves 1910, valve tips 1920 (straighttip in FIG. 19B, bent tip in FIG. 19A, tip block 1930, valve block 1940,valve block assembly 1950, fluid lines 1960, and fluid manifold 1970,fluid reservoir 1980, and pressure system are not shown.

In a preferred embodiment, four linear arrays of 12 valves (banks) eachare placed side by side. The valves and dispenser tips in each of thesearrays are spaced 6 mm apart and each array is staggered 1.5 mm inrelation to each other. The tips of the valves are configured to sharethe same fluid path configuration and align together into a linear arrayof 48 tips spaced 1.5 mm apart. The tips from a single bank of valvesare spaced 6 mm apart and are arranged as every fourth tip in the 48 tiparray Another embodiment uses larger valves that would require largerspacing between each valve. The tips in this embodiment would useflexible tubing to connect the valve and tips.

Each valve can be individually controlled to allow different patterns ofdispensing. Though designed to dispense into a 48 well per column plate,this array could also dispense into other plate configurations. Forexample, 864-well plates (24 by 36 wells), 384-well plates (16 by 24wells) or 96-well plates (8 by 12 wells) are compatible by using everysecond, third or sixth valve, respectively. If the plate can be moved inthe Y as well as the X direction, then each 48 valve linear array candispense up to four different reagents by plumbing a different reagentinto each of the four banks of 12 valves. Moving the plate in both the Xand Y directions allows each well to align with a valve tip from eachbank. If the plate can only be moved in the X direction, then thefollowing dispensing arrangements are possible with a 48-dispenser array(see FIG. 19).

96-Well Plate (6.5 mm diameter wells) Multiple Reagent Mode

A single 48 valve linear array can deliver up to four different reagentsinto a 96-well plate since a single bank can deliver into each well.Each bank is plumbed to receive a different reagent. Plate can besecured properly in the Y direction for each reagent and the plate doesnot need to move in the Y direction during the dispensing of that onereagent.

384 Well Plate (3.4 mm diameter wells) Single Reagent Mode

Valves from each bank must be used in order to dispense into each wellof a 384-well plate; therefore only one reagent can be dispensed fromthe 48 tip array.

864-Well Plate (2.0 mm diameter wells)

Each well can be accessed by using either banks 1 and 3 or banks 2 and4. This allows two different reagents to be delivered if the plate isaligned properly in the Y direction for each reagent.

In operation, the linear array of dispensers can be positioned over ahigh density plate, e.g., 48 by 72-well plate. The wells in this plateare spaced identically to the dispensers (1.5 mm apart). The dispensersare activated and a reagent is dispensed simultaneously into each wellin one column. The plate is then moved over one column and thedispensers are again activated. This is repeated over the entire plate.The amount dispensed is controlled by the valve opening time and thepressure feeding the reagent to the valves (other factors also controldispensed volumes, particularly restrictions to flow). Variable amountscan be dispensed into each well by controlling the timing of valveopening and the dispensation pattern across the linear array of valves.Each dispenser of the linear array can be individually controlled viasoftware.

The entire fluid path can be flushed clean by first purging the fourfluid manifolds, followed by each of the 48 valves. Three 3-way valves(two on the input side and one on the output side of the fluidmanifolds) will allow flushing with a wash liquid and with air. Thedevice can be cleaned between reagent changes and for long-term inactiveperiods. The valve assembly can be designed in a modular form in orderto facilitate replacement and repair of single valve/tip componentsand/or whole banks of valves. The fluid path dead space is preferablydesigned to minimize flush out volumes.

In another embodiment, the dispenser tip pitch can be modulated via acam shaft mechanism which enables on-the-fly control of dispenser tipspacing.

In another embodiment, each valve array can contain a different reagentand address the dispensation requirements of the assay by additionalpositioning movements under the linear array dispenser.

All publications, including patent documents and scientific articles,referred to in this application are incorporated by reference in theirentirety for all purposes to the same extent as if each individualpublication were individually incorporated by reference.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

We claim:
 1. A database comprising chemical activity data forcomputational analysis of chemical structure and activity againsttargets, comprising data of the activity of discrete drug candidatecompounds, said data stored on a computer readable storage media, andsaid data generated by using a method of screening drug candidatecompounds for activity, said method comprising: storing a set of drugcandidate compounds in addressable wells; programming a chemical storageand retrieval module containing said drug candidate compounds for theselection of a subset of said drug candidate compounds; removing saidsubset of said addressable chemical wells from sad chemical storagemodule with an automated robotic retriever; delivering said removedsubset of addressable chemical wells to an automated transport pathway;delivering said removed subset of addressable chemical wells to anautomated liquid handler; retrieving said programmed subset of drugcandidate compounds with said liquid handler by retrieving compoundsfrom less than all, of said removed subset of addressable chemicalwells; screening said selected subset of drug candidate compounds foractivity; and generating data related to the result of said screening.2. The database of claim 1, said method additionally comprisingreturning said selected subset of chemical compounds to said addressablechemical storage and retrieval module via said automated transportpathway.
 3. The database of claim 1, wherein said removing comprisesretrieving said selected set of chemical compounds in a first order ofselection, and wherein said step of delivering said selected subset ofdrug candidate compounds to said automated liquid handler comprisesdelivering said subset of drug candidate compounds in a second order ofselection different from said first order of selection.
 4. The databaseof claim 3, wherein said removing and delivering comprises retrievingand delivering multi-well plates containing said subset of chemicalcompounds.
 5. The database of claim 4, said method additionallycomprising stacking said multi-well plates prior to delivering saidmulti-well plates.
 6. The database of claim 1, wherein said transportpathway comprises at least two parallel lanes; and wherein at least oneof said lanes is configured to provide bidirectional transport of saidchemical compounds.
 7. A method of making a database of chemicalactivity data comprising: storing a set of drug candidate compounds inaddressable wells; programming a chemical storage and retrieval modulecontaining said drug candidate compounds for the selection of a subsetof said drug candidate compounds; removing said subset of saidaddressable chemical wells from sad chemical storage module with anautomated robotic retriever; delivering said removed subset ofaddressable chemical wells to an automated transport pathway; deliveringsaid removed subset of addressable chemical wells to an automated liquidhandler; retrieving said programmed subset of drug candidate compoundswith said liquid handler by retrieving compounds from less than all, ofsaid removed subset of addressable chemical wells; screening saidselected subset of drug candidate compounds for activity; generatingdata related to the result of said screening; and storing said activitydata collected by said reaction module on a computer readable storagemedium.
 8. The method of claim 7, wherein said transport pathwaycomprises at least two parallel lanes; and wherein at least one of saidlanes is configured to provide bidirectional transport of said chemicalcompounds.